Abstruse

An allelic series of mutations is an extremely valuable genetic resource for understanding cistron office. Here we describe eight mutant alleles at the Steel (Sl) locus of mice that were induced with N-ethyl-N-nitrosourea (ENU). The production of the Sl locus is Kit ligand (or Kitl; also known every bit mast jail cell growth factor, stem cell cistron, and Steel cistron), which is a member of the helical cytokine superfamily and is the ligand for the Kit receptor tyrosine kinase. Seven of the eight ENU-induced KitlSl alleles, of which five cause missense mutations, one causes a nonsense mutation and exon skipping, and one affects a splice site, were found to contain bespeak mutations in Kitl. Interestingly, each of the five missense mutations affects residues that are within, or very virtually, conserved α-helical domains of Kitl. These ENU-induced mutants should provide of import information on structural requirements for function of Kitl and other helical cytokines.

THE ligand for Kit, a blazon Iii receptor tyrosine kinase (RTK), is Kitl, which is required for the survival, proliferation, and differentiation of hematopoietic cells, germ cells, and melanocytes (reviewed by Besmeret al. 1993; Levet al. 1994). Although sequence similarities accept allowed assignment of Kit to the platelet-derived growth cistron receptor, β-polypeptide (Pdgfrb) family of RTKs, Kitl does not have pregnant sequence similarities to whatever other growth factor. However, Kitl was predicted to have structural similarities to colony-stimulating factor 1 (Csf1) and fms-like tyrosine kinase 3 ligand (Flt3l) on the ground of limited sequence similarities ( Bazan 1991; Hannumet al. 1994). Recent crystallographic studies have confirmed that Kitl ( Jianget al. 2000; Zhanget al. 2000), Csf1 ( Panditet al. 1992), and Flt3l ( Savvideset al. 2000) share a common structure and are members of the curt-concatenation subgroup of helical cytokines. Interestingly, Kitl, Csf1, and Flt3l are the only helical cytokines that are ligands for members of the Pdgfrb family. Other helical cytokines, such as growth hormone, erythropoietin, and interleukin-2, are ligands for type I or II cytokine receptors, which differ from RTKs in that they lack intrinsic kinase activity. Furthermore, ligands for other members of the Pdgfr family unit, such as platelet-derived growth factor (Pdgf) and vascular endothelial growth factor (Vegf), form an entirely different kind of structure called a cystine knot. Recent studies propose that the Kitl/Csf1/Flt3l grouping shares more than functional properties with the structurally different Pdgf/Vegf group than with the structurally more than similar helical cytokine group ( Jianget al. 2000; Savvideset al. 2000).

Different biologically active isoforms of Kitl occur as either membrane-anchored proteins or soluble proteins and result from alternative RNA splicing and post-translational processing ( Flanaganet al. 1991; Huanget al. 1992). Alternative splicing produces ii Kitl mRNAs (encounter below) that differ by the presence or absence of exon 6, which contains the primary site for proteolytic cleavage. The master translation products of both Kitl mRNAs contain a 25-amino-acid (aa) signal sequence at the N terminus that is not institute in the mature forms of the proteins ( Andersonet al. 1990; Martinet al. 1990). The Kitl mRNA containing exon 6 [(+) E6, also called KL-1 ( Huanget al. 1992)] encodes a 248-aa transmembrane precursor that produces a soluble isoform (S-Kitl) of 165 aa when candy at the principal cleavage site. The Kitl mRNA lacking exon 6 [(-) E6, also chosen KL-2 ( Huanget al. 1992)] encodes a 220-aa transmembrane poly peptide that lacks the chief cleavage site and is predominantly membrane bound (MB-Kitl). In the absenteeism of the chief cleavage site, a secondary cleavage site in exon 7 of mouse Kitl is utilized, causing release of a second S-Kitl isoform ( Majumdaret al. 1994). Both S-Kitl and MB-Kitl class noncovalently linked dimers ( Arakawaet al. 1991; Hsuet al. 1997; Tajimaet al. 1998) and are heavily glycosylated at both Due north- and O-linked sites (Luet al. 1991, 1992; Huanget al. 1992). Although in vitro assays have demonstrated that Southward-Kitl and MB-Kitl are biologically active, MB-Kitl promotes a more persistent activation of Kit than does S-Kitl ( Miyazawaet al. 1995).

Despite great involvement in the biological activities of Kitl, relatively picayune is known of its sequence requirements for function. But three studies of Kitl structure-function relationships in cultured cells have been reported ( Nishikawaet al. 1992; Langleyet al. 1994; Matouset al. 1996). Nishikawaet al. (1992) synthetic a series of C-final deletions and showed that hematopoietic progenitor cell growth in vitro requires only the outset 142 aa of S-Kitl while minimal growth was observed with 133 aa of S-Kitl ( Nishikawaet al. 1992). The importance of three of the 4 α-helical domains to Kitl part was suggested by analysis of mouse-human being chimeras of Due south-Kitl ( Matouset al. 1996). These authors showed farther that substitution of four aa in the 4th helical domain of S-Kitl abolished Kit binding and biological activeness in vitro. While these studies have provided useful information on structural requirements for Kitl role in vitro, they cannot reproduce the intricacies of cell-cell interactions in intact animals.

An allelic series of mutations is a powerful genetic resource for understanding cistron role. In mice, Kitl is encoded past the Sl locus and at that place are at least eighty different KitlSl mutant alleles ( MouseGenomeDatabase 2002; MutantMouseDatabase 2002). Previous studies accept identified various molecular alterations in a number of KitlSl mutant alleles. The about common blazon of KitlSl mutation removes the unabridged Kitl coding region and ranges in size from ∼120-kb deletions to very large, cytogenetically visible deletions ( Cattanachet al. 1993; Bedellet al. 1996a). Other KitlSl alleles (KitlSl-pan and KitlSl-con ) comprise chromosomal rearrangements that exert tissue-specific effects on Kitl mRNA expression, but leave intact the Kitl coding region ( Huanget al. 1993; Bedellet al. 1995). Previous studies have also identified intragenic mutations that affect the Kitl coding region in five KitlSl alleles. The KitlSl-d allele contains an intragenic deletion and potentially encodes a nearly normal South-Kitl but completely lacks MB-Kitl ( Brannanet al. 1991; Flanaganet al. 1991). The KitlSl-17H allele contains a point mutation that affects splicing such that a MB-Kitl with an abnormal cytoplasmic domain is encoded simply the mutation is non expected to touch on production of Southward-Kitl ( Brannanet al. 1992). Interestingly, contempo evidence suggests that KitlSl-17H is mislocalized to the apical compartment, rather than to the baso-lateral compartment, of polarized cells in embryos and that this mislocalization affects melanoblast development and postnatal coat pigmentation ( Wehrle-Haller and Imhof 2001). In the KitlSl-1Neu mutant, the 25-aa signal sequence and an additional 26 aa of the Kitl Due north terminus are missing and in the KitlSl-2Neu mutant merely 96 aa of the normal 165 aa of mature South-Kitl, plus ii additional aa, are present ( Grawet al. 1996). In the KitlSl-3Neu allele, a missense mutation has been identified that causes substitution of serine for asparagine at position 97 of the mature Due south-Kitl and MB-Kitl ( Grawet al. 1997). Although these five KitlSl mutants have provided important information about Kitl office in vivo, a thorough agreement of this signaling molecule would be facilitated by the availability of a larger collection of intragenic KitlSl alleles.

In this commodity we describe viii ethylnitrosourea (ENU)-induced KitlSl mutant alleles, of which seven are shown to comprise point mutations in the Kitl gene. While two of these point mutations are predicted to encode proteins with C-final deletions, five others encode missense substitutions that are located within, or very well-nigh, conserved α-helical domains of Kitl. In the accompanying article ( Rajaramanet al. 2002, this issue) we describe the effects of these mutations on survival, pigmentation, and peripheral blood cells of mice. These mutations should assist provide more information virtually specific residues that are required for Kitl role, as well as nigh function of structurally related cytokines.

MATERIALS AND METHODS

Mice: The new mutations described in this article were generated in specific locus tests conducted at the Oak Ridge National Laboratory using ENU as the mutagen ( Russellet al. 1982). All of the KitlSl mutations except KitlSl-39R were generated in stalk-prison cell spermatogonia past mutagenesis of (101/Rl × C3H/Rl)F1 or (C3H/Rl × 101/Rl)F1 male person mice, followed by mating of mutagenized mice to T-strain females ( Russell 1951). KitlSl-39R was generated in zygotes past mating (B10/Rl × C3H/Rl)Fi females to T-strain males, followed by ENU treatment of impregnated females ( Russellet al. 1988). The T-strain mice used for specific locus tests were homozygous for seven recessive alleles (a, Tyrp1b, Tyrc-ch, Myo5ad, Bmp5se, p, and Ednrbsouth ). Thus, new recessive mutations at these loci would exist revealed amidst first-generation offspring of crosses between mutagenized, or control, (101/F1 × C3H/Rl)Fi and T-strain mice. The progeny were also examined for other visible phenotypes that resulted from new dominant or semidominant mutations. The progeny exhibiting semidominant pigmentation defects were tested for allelism with KitlSl-12R , a known homozygous viable translocation, [T(10D;18D)12Rl], that affects the Kitl locus ( Cacheiro and Russell 1975). Each of the KitlSl alleles used in the nowadays study was made congenic on a common strain background by backcrossing to C3H/Rl mice for >20 generations. Subsequently, each strain was embryo rederived into pathogen-free recipients and is maintained in a pathogen-free colony at the University of Georgia past backcrossing heterozygous mice to inbred C3H/HeNCR mice. KitlSl-gb mice, which carry an ∼120-kb deletion that removes the entire Kitl coding region besides as upstream sequences ( Bedellet al. 1996a), were too maintained on a C3H/HEN CR background and were used in this study.

Total RNA isolation and RT-PCR sequencing assay: Total RNA was prepared from whole embryos or from kidneys and lungs of newborn mice using RNAzol (Tel-Exam, Friendswood, TX) according to the manufacturer's instructions. Full RNA was used as a template for cDNA synthesis using opposite transcription (RT) with random primers and Superscript Ii reverse transcriptase (GIBCO-BRL, Grand Island, NY). The cDNAs were PCR amplified with Taq polymerase (Sigma, St. Louis) using oligonucleotide primers for the Kitl coding region that amplify a product from nucleotide 97 to nucleotide 1067 ( Bedellet al. 1996b). The sequence of the forward primer was v′-CTATCTGCAGCCGCTGCTGG-three′ and the sequence of the reverse primer was 5′-CTGTTACCAGCCACTGTGCG-3′. The (+) E6 and (-) E6 RT-PCR products (encounter Effigy i), which are 970 and 886 bp, respectively, were purified together using Sorcerer PCR Preps Deoxyribonucleic acid purification system (Promega, Madison, WI), and both DNA strands were directly sequenced using automated DNA sequencing. Two sets of primers were used for sequencing; the start prepare corresponds to sequences nested inside the RT-PCR primers and provides sequence for both (+) E6 and (-) E6 RT-PCR templates, while the second ready corresponds to sequences located within exon half dozen and is therefore specific for (+) E6 templates. Nucleotide sequences were aligned and compared using Sequencher software (Gene Codes, Ann Arbor, MI).

Figure 1.

—Analysis of RT-PCR products and genomic sequences of wild-type and mutant Kitl alleles. Schematic representation of alternatively spliced cDNAs from wild type (A), KitlSl-36R mutant (B), and KitlSl-42R mutant (C). In A-C, the numbered rectangular boxes indicate exons, the shaded boxes are coding exons, the open boxes are noncoding exons, and the transmembrane domain of Kitl is represented as a box with vertical lines. (A) Wild-type cDNAs. The (+) E6 transcript encodes a 248-aa precursor protein that is proteolytically cleaved at the site indicated by the arrowhead to produce S-Kitl. The (-) E6 transcript lacks 84 nt and encodes MB-Kitl. (B) KitlSl-36R cDNAs. The asterisk indicates a nonsense mutation at codon 147 and the box with diagonal lines in the (-) E5, (-) E6 cDNA indicates 25 out-of-frame sequences that result from exon skipping. These out-of-frame residues are GlyLysProGlnSerProLeuLysThrArgAlaTyrAsnGlyGlnProTrpHisCysArgLeuSerPheArgLeu. (C) KitlSl-42R cDNAs contain an 8-bp insertion at the junction of exons 4 and 5. (D) The 8-bp insertion in KitlSl-42R cDNA (white letters on black background) causes an insertion of 2 aa followed by a termination codon (Ter). The codon numbers are indicated, with inserted codons marked with an asterisk. (E) KitlSl-42R genomic DNA contains a T → C transition in the 5′ splice donor site of intron 4. The dashed line and lowercase letters represent intron sequences and the solid line and uppercase letters represent exon sequences. Arrows indicate the primers used to amplify and sequence this region.

—Analysis of RT-PCR products and genomic sequences of wild-type and mutant Kitl alleles. Schematic representation of alternatively spliced cDNAs from wild type (A), KitlSl-36R mutant (B), and KitlSl-42R mutant (C). In A-C, the numbered rectangular boxes indicate exons, the shaded boxes are coding exons, the open up boxes are noncoding exons, and the transmembrane domain of Kitl is represented as a box with vertical lines. (A) Wild-type cDNAs. The (+) E6 transcript encodes a 248-aa precursor protein that is proteolytically cleaved at the site indicated past the arrowhead to produce Due south-Kitl. The (-) E6 transcript lacks 84 nt and encodes MB-Kitl. (B) KitlSl-36R cDNAs. The asterisk indicates a nonsense mutation at codon 147 and the box with diagonal lines in the (-) E5, (-) E6 cDNA indicates 25 out-of-frame sequences that consequence from exon skipping. These out-of-frame residues are GlyLysProGlnSerProLeuLysThrArgAlaTyrAsnGlyGlnProTrpHisCysArgLeuSerPheArgLeu. (C) KitlSl-42R cDNAs contain an 8-bp insertion at the junction of exons iv and five. (D) The 8-bp insertion in KitlSl-42R cDNA (white letters on black background) causes an insertion of 2 aa followed by a termination codon (Ter). The codon numbers are indicated, with inserted codons marked with an asterisk. (Due east) KitlSl-42R genomic Dna contains a T → C transition in the five′ splice donor site of intron four. The dashed line and lowercase messages represent intron sequences and the solid line and capital letter letters correspond exon sequences. Arrows indicate the primers used to amplify and sequence this region.

Figure 1.

—Analysis of RT-PCR products and genomic sequences of wild-type and mutant Kitl alleles. Schematic representation of alternatively spliced cDNAs from wild type (A), KitlSl-36R mutant (B), and KitlSl-42R mutant (C). In A-C, the numbered rectangular boxes indicate exons, the shaded boxes are coding exons, the open boxes are noncoding exons, and the transmembrane domain of Kitl is represented as a box with vertical lines. (A) Wild-type cDNAs. The (+) E6 transcript encodes a 248-aa precursor protein that is proteolytically cleaved at the site indicated by the arrowhead to produce S-Kitl. The (-) E6 transcript lacks 84 nt and encodes MB-Kitl. (B) KitlSl-36R cDNAs. The asterisk indicates a nonsense mutation at codon 147 and the box with diagonal lines in the (-) E5, (-) E6 cDNA indicates 25 out-of-frame sequences that result from exon skipping. These out-of-frame residues are GlyLysProGlnSerProLeuLysThrArgAlaTyrAsnGlyGlnProTrpHisCysArgLeuSerPheArgLeu. (C) KitlSl-42R cDNAs contain an 8-bp insertion at the junction of exons 4 and 5. (D) The 8-bp insertion in KitlSl-42R cDNA (white letters on black background) causes an insertion of 2 aa followed by a termination codon (Ter). The codon numbers are indicated, with inserted codons marked with an asterisk. (E) KitlSl-42R genomic DNA contains a T → C transition in the 5′ splice donor site of intron 4. The dashed line and lowercase letters represent intron sequences and the solid line and uppercase letters represent exon sequences. Arrows indicate the primers used to amplify and sequence this region.

—Assay of RT-PCR products and genomic sequences of wild-type and mutant Kitl alleles. Schematic representation of alternatively spliced cDNAs from wild type (A), KitlSl-36R mutant (B), and KitlSl-42R mutant (C). In A-C, the numbered rectangular boxes point exons, the shaded boxes are coding exons, the open up boxes are noncoding exons, and the transmembrane domain of Kitl is represented as a box with vertical lines. (A) Wild-type cDNAs. The (+) E6 transcript encodes a 248-aa precursor protein that is proteolytically broken at the site indicated by the arrowhead to produce S-Kitl. The (-) E6 transcript lacks 84 nt and encodes MB-Kitl. (B) KitlSl-36R cDNAs. The asterisk indicates a nonsense mutation at codon 147 and the box with diagonal lines in the (-) E5, (-) E6 cDNA indicates 25 out-of-frame sequences that result from exon skipping. These out-of-frame residues are GlyLysProGlnSerProLeuLysThrArgAlaTyrAsnGlyGlnProTrpHisCysArgLeuSerPheArgLeu. (C) KitlSl-42R cDNAs contain an 8-bp insertion at the junction of exons four and 5. (D) The eight-bp insertion in KitlSl-42R cDNA (white letters on black groundwork) causes an insertion of 2 aa followed by a termination codon (Ter). The codon numbers are indicated, with inserted codons marked with an asterisk. (E) KitlSl-42R genomic Dna contains a T → C transition in the 5′ splice donor site of intron 4. The dashed line and lowercase messages represent intron sequences and the solid line and capital letters correspond exon sequences. Arrows bespeak the primers used to amplify and sequence this region.

Cloning of Kitl cDNAs: The Kitl coding region of each mutant allele was amplified using primers coordinating to those described above except that they contained restriction sites for the enzymes BamHowdy and PstI. PCR was performed using High Allegiance polymerase (Boehringer Mannheim, Indianapolis). The resulting PCR products were purified equally described to a higher place, digested with BamHowdy and PstI, and cloned into BamHow-do-you-do and PstI sites of Bluescript plasmid (Stratagene, La Jolla, CA). For each mutant allele, two contained clones of the (+) E6 coding region and the (-) E6 coding region were isolated and sequenced using an automatic sequencer.

Northern absorb analysis: Poly(A)+ RNA was prepared from the kidney and lung tissues of wild-blazon and homozygous mutant newborn mice using the Micro-Fast Rail 2.0 kit (Invitrogen, Carlsbad, CA). For Northern blot analysis, the RNAs were electrophoresed through a 1% agarose/vii% formaldehyde gel and transferred to nylon membranes (Boehringer Mannheim). The blot was probed with a digoxygenin (DIG)-labeled antisense riboprobe for the Kitl coding region synthesized from linearized SCF4.one (Kitl) plasmid ( Andersonet al. 1990) as template and reagents from the DIG RNA labeling kit (Boehringer Mannheim). Hybridization, washes, and detection were done according to the manufacturer'south instructions. Afterwards stripping, the same blots were reprobed with a DIG-labeled antisense actin RNA to ostend uniform loading of sample RNAs.

RESULTS

Molecular genetic analysis: All of the new KitlSl mutant alleles in this study were generated following ENU treatment of F1 mice. Although point mutations are the most mutual blazon of sequence alterations observed in germline mutations of mice following ENU treatment ( Noveroskeet al. 2000), other types of genomic alterations are possible. To examine the structural integrity of the Kitl cistron in each of the mutant alleles, Southern absorb analysis of genomic Deoxyribonucleic acid prepared from tissues of mice heterozygous for each mutant allele was performed. The probes used for these studies were a Kitl cDNA that encompasses the entire coding region, a cDNA that encompasses the iii′ untranslated region of Kitl, and a genomic fragment from the v′-flanking region of Kitl ( Bedellet al. 1996b). Together, these probes represent ∼forty kb of genomic Dna. The results point that there are no major structural alterations in the Kitl coding region and in flanking sequences in any of the ENU-induced alleles except KitlSl-25R (data non shown). Analysis of the KitlSl-25R allele revealed multiple brake fragment length polymorphisms (RFLPs) that are consistent with intragenic rearrangements or deletions in the Kitl cistron (data not shown).

TABLE 1

Summary of mutations in Kitl sequences of ENU-induced KitlSl alleles

Kitl
allele a 		Chromosome
of origin b 		Kitl sequence
alteration c 		Result on Kitl d
Sl-30R 101 T325G Missense mutation (L18R)
Sl-31R C3H C340T Missense mutation (P23L)
Sl-22R C3H T433C Missense mutation (L54P)
Sl-28R 101 T626A Missense mutation (I118N)
Sl-42R 101 T → C (five′ splice site) Truncation (96 aa + 2 aa)
Sl-39R B10 C637T Missense mutation (S122F)
Sl-36R C3H G711T Nonsense mutation (E147Ter), exon skipping (96 aa + 25 aa)
Sl-25R 101 Rearrangement or deletion ND
Kitl
allele a 		Chromosome
of origin b 		Kitl sequence
alteration c 		Effect on Kitl d
Sl-30R 101 T325G Missense mutation (L18R)
Sl-31R C3H C340T Missense mutation (P23L)
Sl-22R C3H T433C Missense mutation (L54P)
Sl-28R 101 T626A Missense mutation (I118N)
Sl-42R 101 T → C (5′ splice site) Truncation (96 aa + two aa)
Sl-39R B10 C637T Missense mutation (S122F)
Sl-36R C3H G711T Nonsense mutation (E147Ter), exon skipping (96 aa + 25 aa)
Sl-25R 101 Rearrangement or deletion ND

ND, non determined.

a

All mutants except KitlSl-39R were generated in spermatogonial stem cells by ENU treatment of (101/Rl × C3H/Rl)Fi or (C3H/Rl × 101/Rl)F1 males followed past breeding to T-strain mice ( Russell 1951; Russellet al. 1982). The KitlSl-39R mutation was induced in zygotes resulting from ENU treatment of (B10/Rl × C3H/Rl)Fone females that had mated with T males ( Russellet al. 1988).

b

Each mutant strain is congenic on the C3H strain. Southern blots of Deoxyribonucleic acid from each mutant were hybridized with Kitl cDNA and RFLP analysis of the Kitl factor was used to determine the chromosomal origin of each mutation.

c

The nucleotide numbering for Kitl is from Bedellet al. (1996b; GenBank accession no. U44725).

d

The amino acid numbering is for the processed form of Kitl, which lacks the 25-aa signal sequence.

Table 1

Summary of mutations in Kitl sequences of ENU-induced KitlSl alleles

Kitl
allele a 		Chromosome
of origin b 		Kitl sequence
amending c 		Effect on Kitl d
Sl-30R 101 T325G Missense mutation (L18R)
Sl-31R C3H C340T Missense mutation (P23L)
Sl-22R C3H T433C Missense mutation (L54P)
Sl-28R 101 T626A Missense mutation (I118N)
Sl-42R 101 T → C (5′ splice site) Truncation (96 aa + two aa)
Sl-39R B10 C637T Missense mutation (S122F)
Sl-36R C3H G711T Nonsense mutation (E147Ter), exon skipping (96 aa + 25 aa)
Sl-25R 101 Rearrangement or deletion ND
Kitl
allele a 		Chromosome
of origin b 		Kitl sequence
alteration c 		Effect on Kitl d
Sl-30R 101 T325G Missense mutation (L18R)
Sl-31R C3H C340T Missense mutation (P23L)
Sl-22R C3H T433C Missense mutation (L54P)
Sl-28R 101 T626A Missense mutation (I118N)
Sl-42R 101 T → C (v′ splice site) Truncation (96 aa + 2 aa)
Sl-39R B10 C637T Missense mutation (S122F)
Sl-36R C3H G711T Nonsense mutation (E147Ter), exon skipping (96 aa + 25 aa)
Sl-25R 101 Rearrangement or deletion ND

ND, non determined.

a

All mutants except KitlSl-39R were generated in spermatogonial stalk cells by ENU handling of (101/Rl × C3H/Rl)F1 or (C3H/Rl × 101/Rl)F1 males followed by breeding to T-strain mice ( Russell 1951; Russellet al. 1982). The KitlSl-39R mutation was induced in zygotes resulting from ENU handling of (B10/Rl × C3H/Rl)Fone females that had mated with T males ( Russellet al. 1988).

b

Each mutant strain is congenic on the C3H strain. Southern blots of Deoxyribonucleic acid from each mutant were hybridized with Kitl cDNA and RFLP analysis of the Kitl gene was used to determine the chromosomal origin of each mutation.

c

The nucleotide numbering for Kitl is from Bedellet al. (1996b; GenBank accession no. U44725).

d

The amino acid numbering is for the processed grade of Kitl, which lacks the 25-aa signal sequence.

Since each strain is congenic on C3H, comparison of RFLPs present in the parental strains used for mutagenesis with those of heterozygous mice revealed the chromosome of origin of each mutation (see Tabular array 1). Of the seven mutations induced in (101/Rl × C3H/Rl)F1 or (C3H/Rl × 101/Rl)F1 mice, the KitlSl-30R, KitlSl-42R, KitlSl-28R , and KitlSl-25R mutations occurred on 101/Rl chromosomes while the KitlSl-31R, KitlSl-22R , and KitlSl-36R mutations occurred on C3H/Rl chromosomes. The KitlSl-39R mutation, which was generated from (B10/Rl × C3H/Rl)F1 zygotes, occurred on the B10/Rl chromosome.

To examine the integrity of the Kitl coding region of each allele, total RNA from tissues of homozygous embryos or newborn mice was prepared followed by RT-PCR and nucleotide sequencing of Kitl sequences. The oligonucleotide primers used for RT-PCR were designed such that the entire coding region of Kitl would be amplified. Ii RT-PCR products would be expected from wild-blazon tissues (meet Effigy 1A): (+) E6, which is 970 bp and represents the mRNA encoding S-Kitl, and (-) E6, which is 886 bp and represents the alternatively spliced mRNA encoding MB-Kitl. Considering all coding exons are located in the amplified region, this distension strategy should allow detection of any defects in mRNA splicing that affect the Kitl coding region. Post-obit agarose gel electrophoresis, RT-PCR products of normal size were observed in samples from the KitlSl-22R, KitlSl-28R, KitlSl-30R, KitlSl-31R, KitlSl-39R , and KitlSl-42R tissues. Withal, from KitlSl-36R tissues, an abnormally sized production of 645 bp was observed in add-on to the expected 970- and 886-bp products in KitlSl-36R (see Figure 1B and below). Multiple RT-PCR products of aberrant size, which are consistent with intragenic genomic alterations and abnormal splicing, were observed in KitlSl-25R (not shown). Considering of the complication of these products and because the KitlSl-25R allele behaves as a nada allele (data not shown), the KitlSl-25R RT-PCR products were non characterized further.

Nucleotide sequencing of RT-PCR products was performed for all alleles except KitlSl-25R . RT-PCR products were generated using Taq polymerase then purified and used directly equally templates for nucleotide sequencing. Nucleotide sequence alterations observed with these Taq-generated products were confirmed past sequencing of at least ii cloned RT-PCR products that were generated using High Allegiance polymerase. Using this strategy, nosotros identified a point mutation that affects the Kitl coding region in each of the seven alleles sequenced (Table 1). Each of the sequence alterations observed is consistent with previously described alterations created by treatment with ENU (reviewed past Noveroskeet al. 2000). These mutations are described in more detail (meet beneath), and the Kitl sequences affected in each of these mutant alleles are illustrated in Figure 2.

Figure 2.

—Effects of seven ENU-induced KitlSl mutations on the primary structure of Kitl. A schematic of the protein structure for the seven mutations is shown. The symbols used are as follows: open box, signal sequence; solid boxes αA, αB, αC, and αD, α-helical domains; boxes with horizontal lines, β-sheets; box with diagonal lines, alternately spliced exon 6 sequences; box with vertical lines, transmembrane domain; arrowhead, proteolytic cleavage site; solid ovals, dimer interface; solid circle with line, N-linked glycosylation sites. The positions of the missense mutations are shown above the protein schematic and the sites of truncation predicted for the KitlSl-36R and KitlSl-42R mutants are shown below the protein schematic. For KitlSl-42R, the aberrantly spliced KitlSl-42R cDNA (see Figure 1, C and D) is predicted to produce a truncated protein of 96 aa + 2 aa out of frame. For KitlSl-36R-A, the asterisk represents a premature termination codon found in each of the (+) E6 and (-) E6 cDNAs of KitlSl-36R (see Figure 1B); therefore, each of these transcripts is expected to produce a truncated protein of 146 aa. For KitlSl-36R-B, the (-) E5, (-) E6 cDNA of KitlSl-36R (see Figure 1B) is expected to produce a truncated protein of 96 aa + 25 out-of-frame aa.

—Effects of vii ENU-induced KitlSl mutations on the principal structure of Kitl. A schematic of the protein construction for the seven mutations is shown. The symbols used are as follows: open box, indicate sequence; solid boxes αA, αB, αC, and αD, α-helical domains; boxes with horizontal lines, β-sheets; box with diagonal lines, alternately spliced exon vi sequences; box with vertical lines, transmembrane domain; arrowhead, proteolytic cleavage site; solid ovals, dimer interface; solid circle with line, Northward-linked glycosylation sites. The positions of the missense mutations are shown in a higher place the poly peptide schematic and the sites of truncation predicted for the KitlSl-36R and KitlSl-42R mutants are shown below the protein schematic. For KitlSl-42R, the aberrantly spliced KitlSl-42R cDNA (run into Figure i, C and D) is predicted to produce a truncated protein of 96 aa + two aa out of frame. For KitlSl-36R-A, the asterisk represents a premature termination codon institute in each of the (+) E6 and (-) E6 cDNAs of KitlSl-36R (encounter Figure 1B); therefore, each of these transcripts is expected to produce a truncated protein of 146 aa. For KitlSl-36R-B, the (-) E5, (-) E6 cDNA of KitlSl-36R (see Figure 1B) is expected to produce a truncated protein of 96 aa + 25 out-of-frame aa.

Effigy 2.

—Effects of seven ENU-induced KitlSl mutations on the primary structure of Kitl. A schematic of the protein structure for the seven mutations is shown. The symbols used are as follows: open box, signal sequence; solid boxes αA, αB, αC, and αD, α-helical domains; boxes with horizontal lines, β-sheets; box with diagonal lines, alternately spliced exon 6 sequences; box with vertical lines, transmembrane domain; arrowhead, proteolytic cleavage site; solid ovals, dimer interface; solid circle with line, N-linked glycosylation sites. The positions of the missense mutations are shown above the protein schematic and the sites of truncation predicted for the KitlSl-36R and KitlSl-42R mutants are shown below the protein schematic. For KitlSl-42R, the aberrantly spliced KitlSl-42R cDNA (see Figure 1, C and D) is predicted to produce a truncated protein of 96 aa + 2 aa out of frame. For KitlSl-36R-A, the asterisk represents a premature termination codon found in each of the (+) E6 and (-) E6 cDNAs of KitlSl-36R (see Figure 1B); therefore, each of these transcripts is expected to produce a truncated protein of 146 aa. For KitlSl-36R-B, the (-) E5, (-) E6 cDNA of KitlSl-36R (see Figure 1B) is expected to produce a truncated protein of 96 aa + 25 out-of-frame aa.

—Effects of vii ENU-induced KitlSl mutations on the main structure of Kitl. A schematic of the poly peptide construction for the 7 mutations is shown. The symbols used are as follows: open box, signal sequence; solid boxes αA, αB, αC, and αD, α-helical domains; boxes with horizontal lines, β-sheets; box with diagonal lines, alternately spliced exon half-dozen sequences; box with vertical lines, transmembrane domain; arrowhead, proteolytic cleavage site; solid ovals, dimer interface; solid circle with line, North-linked glycosylation sites. The positions of the missense mutations are shown higher up the protein schematic and the sites of truncation predicted for the KitlSl-36R and KitlSl-42R mutants are shown beneath the protein schematic. For KitlSl-42R, the aberrantly spliced KitlSl-42R cDNA (see Figure one, C and D) is predicted to produce a truncated poly peptide of 96 aa + 2 aa out of frame. For KitlSl-36R-A, the asterisk represents a premature termination codon found in each of the (+) E6 and (-) E6 cDNAs of KitlSl-36R (see Figure 1B); therefore, each of these transcripts is expected to produce a truncated protein of 146 aa. For KitlSl-36R-B, the (-) E5, (-) E6 cDNA of KitlSl-36R (see Figure 1B) is expected to produce a truncated protein of 96 aa + 25 out-of-frame aa.

Premature termination and exon skipping in KitlSl-36R : In the KitlSl-36R allele, an abnormal-size RT-PCR production of 645 bp was observed in addition to the normal-size products of 970 and 886 bp (Effigy 1B). Each of these three KitlSl-36R RT-PCR products was sequenced and the results suggest that two truncated S-Kitl isoforms would be expressed from this allele. In both of the 970- and 886-bp RT-PCR products, a G → T transversion in exon v was identified that creates a nonsense mutation in codon 147. Thus, one mutant product of simply 146 aa of Southward-Kitl would exist produced by premature termination of both of the (+) E6 and (-) E6 transcripts (KitlSl-36R-A in Effigy 2). In the 645-bp RT-PCR product, exon iv was plant to be spliced straight to exon 7 and is likely to upshot from abnormal splicing (skipping) of exon five as well as the normal splicing of exon vi (Figure 1B). Sequencing of 10 cloned 645-bp RT-PCR products from KitlSl-36R /KitlSl-36R kidneys revealed that all 10 utilized the normal exon 4-exon 7 splice junctions (not shown). As the upshot of this exon skipping, sequences downstream to the exon four-exon vii splicing junction are out of frame. Thus, a second KitlSl-36R isoform may be produced by exon skipping and is predicted to encode the first 96 aa of S-Kitl with 25 C-concluding aa out of frame (KitlSl-36R-B in Figure 2).

A splicing defect in KitlSl-42R : Sequencing of the KitlSl-42R RT-PCR products revealed an viii-bp insertion at the junction of exon 4 and exon 5 that creates a termination codon after two codons (Figure 1, C and D). As a result, this allele is predicted to encode the first 96 aa of S-Kitl with ii C-final aa out-of-frame (Effigy ii) and differs from the KitlSl-36R-B isoform in only the out-of-frame C-terminal residues. In the KitlSl-42R RT-PCR production, the 8-bp insertion differs by only 1 nt from a similar eight-bp insertion reported in the KitlSl-2Neu allele ( Grawet al. 1996). Because both insertions are located at the junction of exon 4 and exon 5, it was possible that the insertions effect from a splicing defect. To examine this hypothesis, we determined the sequence of the splice donor site of intron 4 from wild-blazon and KitlSl-42R alleles. Intron four of wild-type genomic Deoxyribonucleic acid was amplified using oligonucleotide primers from exons iv and 5. Nucleotide sequencing revealed that the commencement viii nucleotide (nt) of this intron differ from the KitlSl-42R insertion past only 1 nt (meet Effigy 1E). Sequencing of the respective region of PCR-amplified genomic DNA from KitlSl-42R / KitlSl-42R tissue revealed the presence of a T → C substitution in the 5′ splice donor site (run across Effigy 1E). A second 5′ splice donor site (GT) is located just 9 nt 3′ of the commonly used 5′ donor site and is unaffected by the KitlSl-42R mutation (wild type, five′-GTAACTTGGT-3′; mutant, 5′-GCAACTTGGT-three′). Thus, the KitlSl-42R allele contains a point mutation that abolishes the normal splice donor site, resulting in use of a cryptic splice donor site in intron 4. To make up one's mind the efficiency of normal and abnormal splicing events, we performed RT-PCR on mRNA from fetal liver of wild-blazon and KitlSl-42R /KitlSl-42R embryos using primers that flank the viii-bp insertion. The results revealed that in that location were no RT-PCR products of normal size detectable in KitlSl-42R /KitlSl-42R tissue (non shown). Thus, the vast majority of the KitlSl-42R mRNA is aberrantly spliced such that lilliputian or no wild-type mRNA is expressed.

Missense mutations in 5 KitlSl alleles: Each of the five missense mutations affects sequences that are within or near conserved α-helical domains of Kitl (run across Figure 2). To gain insight into the functional importance of residues afflicted past the KitlSl missense mutations, we also examined the conservation of each affected residual in orthologous sequences. Accordingly, the residues afflicted in each mutant are superimposed on a multiple sequence alignment of the processed Southward-Kitl orthologs from nine mammals in Figure iii. In addition, Figure three illustrates some structural aspects of human Kitl (from Jianget al. 2000).

Figure iii.

—Multiple sequence alignment of Kitl orthologs with residues affected in KitlSl missense mutations. S-Kitl sequences were aligned and plotted using Wisconsin Package Version 10.1 (Genetics Computer Group, Madison, WI). GenBank accession numbers are as follows: mouse (Mus musculus), U44725; rat (Rattus norvegicus), AF071204; cow (Bos taurus), D28934; sheep (Ovis aries), U89874; cat (Felis catus), D50833; pig (Sus scrofa), L07786; dog (Canis familiaris), S53329; horse (Equus caballus), AF053498; human (Homo sapiens), M59964. Identical residues are white letters on a black background; different residues are black letters on a white background; and similar residues are black letters on shaded background. Structural features (solid rectangles, α-helical domains; shaded rectangles, β-sheets; solid ovals, dimer interface; ball and stick, N-linked glycosylation sites) are from the published crystal structure of human Kitl (Jiang  et al. 2000). The positions of missense mutations described in this report are shown above the mouse sequence.

—Multiple sequence alignment of Kitl orthologs with residues affected in KitlSl missense mutations. S-Kitl sequences were aligned and plotted using Wisconsin Package Version 10.one (Genetics Computer Group, Madison, WI). GenBank accretion numbers are as follows: mouse (Mus musculus), U44725; rat (Rattus norvegicus), AF071204; cow (Bos taurus), D28934; sheep (Ovis aries), U89874; cat (Felis catus), D50833; pig (Sus scrofa), L07786; canis familiaris (Canis familiaris), S53329; equus caballus (Equus caballus), AF053498; human (Homo sapiens), M59964. Identical residues are white letters on a blackness groundwork; different residues are black letters on a white background; and similar residues are black letters on shaded background. Structural features (solid rectangles, α-helical domains; shaded rectangles, β-sheets; solid ovals, dimer interface; brawl and stick, Northward-linked glycosylation sites) are from the published crystal construction of human Kitl ( Jianget al. 2000). The positions of missense mutations described in this report are shown above the mouse sequence.

Figure iii.

—Multiple sequence alignment of Kitl orthologs with residues affected in KitlSl missense mutations. S-Kitl sequences were aligned and plotted using Wisconsin Package Version 10.1 (Genetics Computer Group, Madison, WI). GenBank accession numbers are as follows: mouse (Mus musculus), U44725; rat (Rattus norvegicus), AF071204; cow (Bos taurus), D28934; sheep (Ovis aries), U89874; cat (Felis catus), D50833; pig (Sus scrofa), L07786; dog (Canis familiaris), S53329; horse (Equus caballus), AF053498; human (Homo sapiens), M59964. Identical residues are white letters on a black background; different residues are black letters on a white background; and similar residues are black letters on shaded background. Structural features (solid rectangles, α-helical domains; shaded rectangles, β-sheets; solid ovals, dimer interface; ball and stick, N-linked glycosylation sites) are from the published crystal structure of human Kitl (Jiang  et al. 2000). The positions of missense mutations described in this report are shown above the mouse sequence.

—Multiple sequence alignment of Kitl orthologs with residues affected in KitlSl missense mutations. S-Kitl sequences were aligned and plotted using Wisconsin Package Version 10.1 (Genetics Figurer Group, Madison, WI). GenBank accession numbers are as follows: mouse (Mus musculus), U44725; rat (Rattus norvegicus), AF071204; cow (Bos taurus), D28934; sheep (Ovis aries), U89874; cat (Felis catus), D50833; pig (Pig), L07786; dog (Dog), S53329; equus caballus (Equus caballus), AF053498; homo (Homo sapiens), M59964. Identical residues are white messages on a blackness background; dissimilar residues are black letters on a white background; and like residues are black messages on shaded background. Structural features (solid rectangles, α-helical domains; shaded rectangles, β-sheets; solid ovals, dimer interface; brawl and stick, N-linked glycosylation sites) are from the published crystal structure of human Kitl ( Jianget al. 2000). The positions of missense mutations described in this report are shown above the mouse sequence.

The KitlSl-30R mutation results from a T325G transversion that causes a L18R missense mutation in αA. The leucine at position eighteen is conserved in all mammalian orthologs and affects a residue buried in the human Kitl dimer (Figure iii). Furthermore, the side-chains of leucine residues are idea to form the hydrophobic inner cadre of α-helices and may be of import for helix packing. Thus, replacement of the positively charged arginine for the hydrophobic, nonpolar leucine is likely to have a significant effect on poly peptide conformation.

The KitlSl-31R mutation results from a C340T transition that causes a P23L missense mutation at the very end of αA. Interestingly, the proline at position 23 in Kitl is conserved in all mammalian orthologs (Figure three) and prolines flanked by polar residues are found frequently at the termini of α-helices ( Gunasekaranet al. 1998). Thus, the P23L replacement in Kitl may impact the termination of αA. Furthermore, this exchange affects a residue at the man Kitl dimer interface ( Jianget al. 2000), suggesting that the KitlSl-31R encoded protein may not be able to dimerize efficiently.

The KitlSl-22R mutation results from a T433C transition that causes a L54P missense mutation in αB. This mutation also affects a DdeI restriction site (CTCAG to CCCAG), and restriction enzyme digestion of PCR-amplified genomic DNA from wild-type and homozygous mutant tissues confirmed the sequence amending identified in RT-PCR products (non shown). Unlike residues affected past the other Kitl missense mutations, the leucine at position 54 is not conserved but is a valine, another hydrophobic, nonpolar amino acrid, in 7 of the nine mammalian orthologs (Figure 3). However, prolines are mostly incompatible with α-helical domains and the presence of the L54P replacement within αB, like the L18R replacement in αA of the KitlS-30R mutant, is probable to have a significant effect on local tertiary structure.

The KitlSl-28R mutation results from a T626A transversion that causes an I118N missense mutation in αD. The isoleucine at position 118 is found in all ix mammalian orthologs (Effigy 3). Exchange of the polar asparagine remainder for the hydrophobic, cached isoleucine residue would exist expected to have a major effect on local construction of that region.

Figure 4.

—Northern blot of Kitl mRNA levels in tissues of KitlSl mutant mice. Poly(A)+ mRNA isolated from kidney and lung of newborn mice was electrophoresed, blotted, and hybridized to digoxigenin-labeled Kitl antisense probe (top) and β-actin antisense probe (bottom). For each lane the allele and genotypes are: lane 1, KitlSl-gb/KitlSl-gb; lane 2, KitlSl-30R/KitlSl-30R; lane 3, KitlSl-31R/KitlSl-31R; lane 4, KitlSl-22R/KitlSl-22R; lane 5, KitlSl-28R/ KitlSl-28R; lane 6, KitlSl-39R/KitlSl-39R; lane 7, KitlSl-42R/KitlSl-42R; lane 8, KitlSl-36R/KitlSl-36R; and lane 9, Kitl+/Kitl+.

—Northern blot of Kitl mRNA levels in tissues of KitlSl mutant mice. Poly(A)+ mRNA isolated from kidney and lung of newborn mice was electrophoresed, blotted, and hybridized to digoxigenin-labeled Kitl antisense probe (summit) and β-actin antisense probe (bottom). For each lane the allele and genotypes are: lane one, KitlSl-gb /KitlSl-gb ; lane 2, KitlSl-30R /KitlSl-30R ; lane 3, KitlSl-31R /KitlSl-31R ; lane 4, KitlSl-22R /KitlSl-22R ; lane v, KitlSl-28R / KitlSl-28R ; lane six, KitlSl-39R /KitlSl-39R ; lane 7, KitlSl-42R /KitlSl-42R ; lane 8, KitlSl-36R /KitlSl-36R ; and lane 9, Kitl +/Kitl +.

Figure four.

—Northern blot of Kitl mRNA levels in tissues of KitlSl mutant mice. Poly(A)+ mRNA isolated from kidney and lung of newborn mice was electrophoresed, blotted, and hybridized to digoxigenin-labeled Kitl antisense probe (top) and β-actin antisense probe (bottom). For each lane the allele and genotypes are: lane 1, KitlSl-gb/KitlSl-gb; lane 2, KitlSl-30R/KitlSl-30R; lane 3, KitlSl-31R/KitlSl-31R; lane 4, KitlSl-22R/KitlSl-22R; lane 5, KitlSl-28R/ KitlSl-28R; lane 6, KitlSl-39R/KitlSl-39R; lane 7, KitlSl-42R/KitlSl-42R; lane 8, KitlSl-36R/KitlSl-36R; and lane 9, Kitl+/Kitl+.

—Northern blot of Kitl mRNA levels in tissues of KitlSl mutant mice. Poly(A)+ mRNA isolated from kidney and lung of newborn mice was electrophoresed, blotted, and hybridized to digoxigenin-labeled Kitl antisense probe (top) and β-actin antisense probe (bottom). For each lane the allele and genotypes are: lane 1, KitlSl-gb /KitlSl-gb ; lane 2, KitlSl-30R /KitlSl-30R ; lane iii, KitlSl-31R /KitlSl-31R ; lane 4, KitlSl-22R /KitlSl-22R ; lane v, KitlSl-28R / KitlSl-28R ; lane 6, KitlSl-39R /KitlSl-39R ; lane 7, KitlSl-42R /KitlSl-42R ; lane viii, KitlSl-36R /KitlSl-36R ; and lane ix, Kitl +/Kitl +.

The KitlSl-39R mutation results from a C637T transition that causes a S122F missense mutation in αD. This mutation abolishes a MboI brake enzyme site (GATC) and creates a Hinf I restriction site (GATTC). These alterations were confirmed using restriction enzyme digestion of RT-PCR products from wild-type and KitlSl-39R tissues (non shown). The serine at position 122 is conserved in all nine mammalian orthologs (Figure 3) and is a office of a recognition sequence for N-linked glycosylation, i.eastward., Asn-X-Ser/Thr. Consequently, the S122F commutation would disrupt i of iv N-linked glycosylation sites identified in mouse Kitl (encounter Figure 2). Although in vitro studies with recombinant S-Kitl accept indicated that glycosylation is not essential for biological action ( Langleyet al. 1994), North-linked glycans are known to facilitate protein folding and conformational maturation ( Helenius 1994; O'Connor and Imperiali 1996). Thus, altered glycosylation of KitlSl-39R -encoded Kitl may contribute to altered role in vivo.

Steady-state levels of mutant Kitl mRNAs: To determine whether whatever of the mutations bear on steady-state levels of Kitl mRNA, Northern absorb analysis of tissues from mice homozygous for each mutation was performed. To ensure that the probe did not cantankerous-hybridize to other mRNAs, a control lane contained RNA prepared from tissues of KitlSl-gb /KitlSl-gb mice, which are homozygous for a deletion that removes the Kitl coding region ( Bedellet al. 1996a). RNAs from at least two mice homozygous for each mutant allele were analyzed in separate Northern blots and each blot independent lanes with RNA from Kitl +/Kitl + and KitlSl-gb /KitlSl-gb mice. A representative Northern blot is shown in Figure 4. Although a pocket-size amount of variation was noted for samples of the same genotype on unlike blots, the combined results from all blots reveal that all mutant alleles except KitlSl-gb limited a Kitl transcript of similar size and affluence to that of the wild-type allele. Because these studies were performed on lung and kidney of newborn mice, nosotros cannot exclude the possibility of tissue-specific or developmental-stage-specific furnishings on Kitl mRNA affluence. Nonetheless, such effects are unlikely. It therefore may be concluded that the major effect of each mutation is on the structure and/or function of the Kitl protein.

Word

In this written report we describe the molecular genetic alterations in eight KitlSl mutant alleles that arose from ENU mutagenesis. One of these alleles (KitlSl-25R ) contains an intragenic rearrangement or deletion; however, it is not clear whether that alteration was induced by ENU treatment or resulted from a coincidental, spontaneous mutation in an ENU-treated mouse. Point mutations were found in seven of the eight KitlSl alleles and were of five types: two A/T → G/C transitions (KitlSl-22R and KitlSl-42R ); two G/C → A/T transitions (KitlSl-31R and KitlSl-39R ); an A/T → T/A transversion (KitlSl-28R ); an A/T → C/G transversion (KitlSl-30R ); and a G/C → T/A transversion (KitlSl-36R ). Such molecular multifariousness is somewhat surprising considering that 82% of the previously characterized ENU-induced germline mutations in the mouse were either A/T → M/C transitions or A/T → T/A transversions ( Noveroskeet al. 2000). The merely previously reported ENU-induced KitlSl mutation was an A/T → T/A transversion in the KitlSl-17H allele ( Brannanet al. 1992). Two aspects of the ENU-induced KitlSl allelic series are noteworthy. Starting time, the KitlSl-36R is only the second Thou/C → T/A transversion reported for an ENU-induced mouse germline mutation ( Noveroskeet al. 2000). This type of mutation has been reported previously only in a Pax6Sey mutant allele ( Hillet al. 1991). Second, the G/C → A/T transition in the KitlSl-39R allele is, to our knowledge, the first characterized mutation that resulted from ENU treatment of zygotes. The vast majority of ENU-induced germline mutations in mice have occurred in spermatogonia and it will be of interest to determine if the mutational spectrum is different betwixt the two types of cells. Together, our observations of KitlSl alleles add to the notion that ENU causes predominantly bespeak mutations in mice ( Markeret al. 1997; Justiceet al. 1999; Noveroskeet al. 2000).

The mutagenesis tests that generated the present mutations were designed to detect recessive mutations at 7 defined loci and visible dominant mutations at other loci ( Russell 1951; Russellet al. 1982). The new KitlSl alleles were detected because they exhibited semidominant pigmentation phenotypes (described in Rajaramanet al. 2002). Including previously reported KitlSl alleles ( Kurodaet al. 1988; Copelandet al. 1990; Brannanet al. 1991, 1992; Cattanachet al. 1993; Bedellet al. 1995, 1996a; Grawet al. 1996, 1997) and unpublished KitlSl alleles ( MouseGenomeDatabase 2002; MutantMouseDatabase 2002), this brings the total number of KitlSl mutant alleles to at to the lowest degree 80. Such a big allelic series exists at but a scattering of other loci in the mouse (see MouseGenomeDatabase 2002; MutantMouseDatabase 2002 and review by Davis and Justice 1998), notably loci included in the specific locus test (a, Tyr1b, Tyrc, Myo5ad, Bmp5se, p, and Ednrbs ), as well as Mitf Mi and KitW . In the KitlSl allelic series, the variation in phenotypic severity and diversity of molecular defects is quite remarkable. Some KitlSl alleles contain deletions that completely remove the Kitl gene, as well as closely linked genes ( Cattanachet al. 1993; Bedellet al. 1996a). While these deletions can be useful for identifying genes in the vicinity of Kitl, they provide little information regarding structural requirements for Kitl function. Hypomorphic alleles, on the other paw, are very valuable because they may let phenotypic analyses at developmental stages afterwards than those reached by null mutants and may reveal more subtle furnishings ( Schumacheret al. 1996). Previously characterized hypomorphic KitlSl alleles include two that touch on expression of Kitl mRNA (KitlSl-pan and KitlSl-con , Bedellet al. 1995), four that affect entire domains of the Kitl protein (KitlSl-d , Brannanet al. 1991 and Flanaganet al. 1991; KitlSl-17H , Brannanet al. 1992; KitlSl-1Neu and KitlSl-2Neu , Grawet al. 1996), and one missense mutation in Kitl (KitlSl-3Neu , Grawet al. 1997). While ii of the KitlSl alleles in the nowadays study besides affect entire domains of Kitl considering of premature termination and abnormal mRNA splicing (KitlSl-36R and KitlSl-42R ), v alleles were found to contain missense mutations (KitlSl-30R, KitlSl-31R, KitlSl-22R, KitlSl-28R , and KitlSl-39R ). These alleles should provide important information on critical residues and domains that are required for Kitl part. In the accompanying article ( Rajaramanet al. 2002) we provide evidence that v of these new alleles (KitlSl-30R, KitlSl-31R, KitlSl-22R, KitlSl-28R , and KitlDue south-42R ) have little or no functional activity for mouse survival or development of peripheral blood cells while two of these alleles are hypomorphic (KitlSl-36R and KitlSl-39R ) for these activities.

A recent compilation of ENU-induced mutations in the mouse has reported that 26% of published germline mutations have splicing errors ( Justiceet al. 1999). Consequent with this observation, we describe two mutations (KitlSl-42R and KitlSl-36R ) that affect Kitl mRNA splicing. Still, the mechanisms by which the abnormal splicing occurs are distinctly different in these mutants. The KitlSl-42R allele contains a point mutation in the normal splice donor site of intron iv (Figure one, D and Eastward) and abolishes normal splicing of that intron. Comparison of the splice donor sequences in 3724 splice sites has revealed that GT are invariant residues at the 5′ end of introns, and at that place is substantial evidence that these nucleotides are essential to right mRNA splicing ( Senapathyet al. 1990). In dissimilarity, nucleotide sequence analysis of RT-PCR products from the KitlSl-36R allele predicts ii molecular defects: the cosmos of a nonsense mutation in exon 5, which is present in both the (+) E6 and (-) E6 culling transcripts, and skipping of exon 5, which generates the (-) E5, (-) E6 cDNA (Figure 1B). Since the get-go written report in 1993 ( Dietzet al. 1993), there have been numerous examples of nonsense-mediated exon skipping ( Valentine 1998). However, the mechanism by which nonsense-mediated exon skipping occurs is currently unknown. Valentine has recently reported that nonsense (and missense) mutations associated with exon skipping often occur in purine-rich sequences that are within thirty bp of exon-intron boundaries and usually involve commutation to a thymidine residue ( Valentine 1998). Consistent with this, the nonsense mutation in KitlSl-36R is created by a G → T transversion that occurs within a ThouAGAAAG sequence (where the underlined G is converted to T in the mutant) that immediately precedes that boundary betwixt exon v and intron five.

Although truncation mutants are useful for understanding the part(s) of dissimilar protein domains, missense mutations are most useful for identifying disquisitional residues that touch structure and office. At present, we exercise not know the mechanism past which the KitlSl missense mutations affect Kitl part. Because our studies advise that the steady-land level of Kitl mRNA expressed by each allele is normal (Figure iv), it is likely that some aspect of Kitl construction or function is affected. Since all of the Kitl missense mutations are within or very near α-helical domains, information technology is very likely that loss of part by these mutants is caused by impaired structural integrity. If the Kitl mutants do in fact have major structural defects, it is possible that the misfolded mutant proteins are retained intracellularly and/or may have increased turnover. Altered glycosylation may also play a role in the KitlSl-39R mutant, as this point mutation affects a site for N-linked glycosylation at Asn 120 (Luet al. 1991, 1992). The last possibility is that the mutant Kitl may not collaborate properly with Kit and may exhibit either quantitative or qualitative differences in binding to and activating the receptor. Farther studies that examine each of the in a higher place aspects of Kitl processing, localization, and binding to Kit by each of the KitlSl mutants volition be necessary.

Acknowledgement

We thank Drs. Neal Copeland and Nancy Jenkins for their back up during the early stages of these experiments and for providing resources for embryo rederivation and Keycharianne Gómez for technical assistance. We likewise give thanks Drs. Eugene Rinchik and David Williams and two anonymous reviewers for their comments on the manuscript. The mutants were generated in enquiry jointly sponsored past the Function of Biological and Environmental Research, USDOE, at the Oak Ridge National Laboratory, managed by UT-Batelle, LLC, nether contract DE-AC05-00OR22725, and by the National Plant of Environmental Health Sciences under interagency understanding no. Y1-ES-8048/0524-I119-A1. The cloth in this manuscript is based on work supported by the National Science Foundation nether grant no. IBN-9728428 and the Academy of Georgia Enquiry Foundation.

Footnotes

Communicating editor: C. Kozak

LITERATURE CITED

Anderson

D M

,

Lyman

Southward D

,

Baird

A

,

Wignall

J M

,

Eisenman

J

 et al. ,

1990

Molecular cloning of mast cell growth factor, a hema-topoietin that is active in both membrane bound and soluble forms

.

Cell

63

:

235

–

243

.

Arakawa

T

,

Yphantis

D A

,

Lary

J W

,

Narhi

L O

,

Lu

H S

 et al. ,

1991

Glycosylated and unglycosylated recombinant-derived man stem cell factors are dimeric and have extensive regular secondary structure

.

J. Biol. Chem.

266

:

18942

–

18948

.

Bazan

J F

,

1991

Genetic and structural homology of stem cell factor and macrophage colony-stimulating factor

.

Cell

65

:

9

–

x

.

Bedell

M A

,

Brannan

C I

,

Evans

E P

,

Copeland

N K

,

Jenkins

N A

 et al. ,

1995

DNA rearrangements located over 100 kb five′ of the Steel (Sl)-coding region in Steel-panda and Steel-contrasted mice deregulate Sl expression and crusade female sterility past disrupting ovarian follicle development

.

Genes Dev.

9

:

455

–

470

.

Bedell

M A

,

Cleveland

L S

,

O'Sullivan

T North

,

Copeland

North 1000

,

Jenkins

N A

,

1996a

Deletion and interallelic complementation analysis of Steel mutant mice

.

Genetics

142

:

935

–

944

.

Bedell

Yard A

,

Copeland

North G

,

Jenkins

N A

,

1996b

Multiple pathways for Steel regulation suggested by genomic and sequence analysis of the murine Steel gene

.

Genetics

142

:

927

–

934

.

Besmer

P

,

Manova

K

,

Duttlinger

R

,

Huang

East J

,

Packer

A

 et al. ,

1993

The kit-ligand (steel factor) and its receptor c-kit/W: pleiotropic roles in gametogenesis and melanogenesis

.

Evolution

(

Suppl.

):

125

–

137

.

Brannan

C I

,

Lyman

Due south D

,

Williams

D E

,

Eisenman

J

,

Anderson

D M

 et al. ,

1991

Steel-Dickie mutation encodes a c-kit ligand defective transmembrane and cytoplasmic domains

.

Proc. Natl. Acad. Sci. United states

88

:

4671

–

4674

.

Brannan

C I

,

Bedell

M A

,

Resnick

J L

,

Eppig

J J

,

Handel

M A

 et al. ,

1992

Developmental abnormalities in Steel17H mice result from a splicing defect in the steel factor cytoplasmic tail

.

Genes Dev.

6

:

1832

–

1842

.

Cacheiro

North Fifty A

,

Russell

L B

,

1975

Evidence that linkage group 4 every bit well equally linkage group X of the mouse are in chromosome ten

.

Genet. Res.

25

:

193

–

195

.

Cattanach

B Chiliad

,

Burtenshaw

M D

,

Rasberry

C

,

Evans

Due east P

,

1993

Large deletions and other gross forms of chromosome imbalance compatible with viability and fertility in the mouse

.

Nat. Genet.

three

:

56

–

61

.

Copeland

Northward Chiliad

,

Gilbert

D J

,

Cho

B C

,

Donovan

P J

,

Jenkins

North A

 et al. ,

1990

Mast cell growth factor maps near the Steel locus on mouse chromosome 10 and is deleted in a number of Steel alleles

.

Prison cell

63

:

175

–

183

.

Davis

A P

,

Justice

M J

,

1998

Mouse alleles: if you've seen i, you haven't seen them all

.

Trends Genet.

14

:

438

–

441

.

Dietz

H C

,

Valle

D

,

Francomano

C A

,

Kendzior

R J

Jr.,

Pyeritz

R Eastward

 et al. ,

1993

The skipping of constitutive exons in vivo induced by nonsense mutations

.

Science

259

:

680

–

683

.

Flanagan

J 1000

,

Chan

D C

,

Leder

P

,

1991

Transmembrane form of the kit ligand growth cistron is determined by alternative splicing and is missing in the Sld mutant

.

Cell

64

:

1025

–

1035

.

Graw

J

,

Loster

J

,

Neuhäuser-Klaus

A

,

Pretsch

W

,

Schmitt-John

T

,

1996

Molecular assay of two new Steel mutations in mice shows a transversion or an insertion

.

Mamm. Genome

7

:

843

–

846

.

Graw

J

,

Neuhäuser-Klaus

A

,

Pretsch

W

,

1997

Detection of a bespeak mutation (A to M) in exon 5 of the murine Mgf gene defines a novel allele at the Steel locus with a weak phenotype

.

Mutat. Res.

382

:

75

–

78

.

Gunasekaran

K

,

Nagarajaram

H A

,

Ramakrishnan

C

,

Balaram

P

,

1998

Stereochemical punctuation marks in protein structures: glycine and proline containing helix stop signals

.

J. Mol. Biol.

275

:

917

–

932

.

Hannum

C

,

Culpepper

J

,

Campbell

D

,

McClanahan

T

,

Zurawski

S

 et al. ,

1994

Ligand for FLT3/FLK2 receptor tyrosine kinase regulates growth of haematopoietic stalk cells and is encoded by variant RNAs

.

Nature

368

:

643

–

648

.

Helenius

A

,

1994

How N-linked oligosaccharides impact glycoprotein folding in the endoplasmic reticulum. Mol. Biol

.

Cell

v

:

253

–

265

.

Hill

R E

,

Favor

J

,

Hogan

B Fifty

,

Ton

C C

,

Saunders

Yard F

 et al. ,

1991

Mouse small eye results from mutations in a paired-like homeobox-containing gene

.

Nature

354

:

522

–

525

.

Hsu

Y R

,

Wu

G One thousand

,

Mendiaz

E A

,

Syed

R

,

Wypych

J

 et al. ,

1997

The bulk of stem cell gene exists as monomer under physiological weather condition. Implications for dimerization mediating biological activeness

.

J. Biol. Chem.

272

:

6406

–

6415

.

Huang

Due east J

,

Nocka

K H

,

Cadet

J

,

Besmer

P

,

1992

Differential expression and processing of two cell associated forms of the kitligand: KL-1 and KL-2. Mol. Biol

.

Cell

iii

:

349

–

362

.

Huang

Due east J

,

Manova

K

,

Packer

A I

,

Sanchez

S

,

Bachvarova

R F

 et al. ,

1993

The murine steel panda mutation affects kit ligand expression and growth of early on ovarian follicles

.

Dev. Biol.

157

:

100

–

109

.

Jiang

X

,

Gurel

O

,

Mendiaz

Due east A

,

Stearns

K W

,

Clogston

C L

 et al. ,

2000

Construction of the agile core of human being stem cell factor and analysis of bounden to its receptor Kit

.

EMBO J.

19

:

3192

–

3203

.

Justice

Yard J

,

Noveroske

J K

,

Weber

J Due south

,

Zheng

B

,

Bradley

A

,

1999

Mouse ENU mutagenesis

.

Hum. Mol. Genet.

8

:

1955

–

1963

.

Kuroda

H

,

Terada

Due north

,

Nakayama

H

,

Matsumoto

K

,

Kitamura

Y

,

1988

Infertility due to growth abort of ovarian follicles in Sl/Sl t mice

.

Dev. Biol.

126

:

71

–

79

.

Langley

M E

,

Mendiaz

E A

,

Liu

N

,

Narhi

L O

,

Zeni

50

 et al. ,

1994

Properties of variant forms of human stem cell gene recombinantly expressed in Escherichia coli

.

Arch. Biochem. Biophys.

311

:

55

–

61

.

Lev

S

,

Blechman

J Thou

,

Givol

D

,

Yarden

Y

,

1994

Steel cistron and c-kit protooncogene: genetic lessons in signal transduction

.

Crit. Rev. Oncog.

five

:

141

–

168

.

Lu

H Southward

,

Clogston

C L

,

Wypych

J

,

Fausset

P R

,

Lauren

S

 et al. ,

1991

Amino acid sequence and mail-translational modification of stem cell factor isolated from buffalo rat liver cell-conditioned medium

.

J. Biol. Chem.

266

:

8102

–

8107

.

Lu

H S

,

Clogston

C L

,

Wypych

J

,

Parker

V P

,

Lee

T D

 et al. ,

1992

Post-translational processing of membrane-associated recombinant homo stem prison cell cistron expressed in Chinese hamster ovary cells

.

Curvation. Biochem. Biophys.

298

:

150

–

158

.

Majumdar

M K

,

Feng

Fifty

,

Medlock

East

,

Toksoz

D

,

Williams

D A

,

1994

Identification and mutation of main and secondary proteolytic cleavage sites in murine stalk cell gene cDNA yields biologically active, cell-associated protein

.

J. Biol. Chem.

269

:

1237

–

1242

.

Marker

P C

,

Seung

1000

,

Bland

A E

,

Russell

L B

,

Kingsley

D M

,

1997

Spectrum of Bmp5 mutations from germline mutagenesis experiments in mice

.

Genetics

145

:

435

–

443

.

Martin

F H

,

Suggs

S Five

,

Langley

K E

,

Lu

H Due south

,

Ting

J

 et al. ,

1990

Primary structure and functional expression of rat and human stem prison cell cistron DNAs

.

Cell

63

:

203

–

211

.

Matous

J Five

,

Langley

Chiliad

,

Kaushansky

G

,

1996

Structure-office relationships of stem cell factor: an analysis based on a serial of man-murine stem cell factor chimera and the mapping of a neutralizing monoclonal antibody

.

Claret

88

:

437

–

444

.

Miyazawa

K

,

Williams

D A

,

Gotoh

A

,

Nishimaki

J

,

Broxmeyer

H E

 et al. ,

1995

Membrane-spring Steel gene induces more than persistent tyrosine kinase activation and longer life span of c-kit cistron-encoded protein than its soluble form

.

Blood

85

:

641

–

649

.

Nishikawa

K

,

Tojo

A

,

Ikebuchi

K

,

Katayama

K

,

Fujii

N

 et al. ,

1992

Deletion mutagenesis of stalk cell factor defines the C-terminal sequences essential for its biological activeness

.

Biochem. Biophys. Res. Commun.

188

:

292

–

297

.

Noveroske

J K

,

Weber

J Southward

,

Justice

M J

,

2000

The mutagenic activity of N-ethyl-North-nitrosourea in the mouse

.

Mamm. Genome

11

:

478

–

483

.

O'Connor

S E

,

Imperiali

B

,

1996

Modulation of protein structure and function by asparagine-linked glycosylation

.

Chem. Biol.

3

:

803

–

812

.

Pandit

J

,

Bohm

A

,

Jancarik

J

,

Halenbeck

R

,

Koths

K

 et al. ,

1992

Three-dimensional construction of dimeric human being recombinant macrophage colony-stimulating factor

.

Science

258

:

1358

–

1362

.

Rajaraman

Southward

,

Davis

W Southward

,

Mahakali-Zama

A

,

Evans

H K

,

Russell

L B

 et al. ,

2002

An allelic serial of mutations in the Kit ligand gene of mice. 2. Effects of ethylnitrosourea-induced Kitl point mutations on survival and peripheral claret cells of KitlSteel mice

.

Genetics

162

:

341

–

353

.

Russell

L B

,

Bangham

J W

,

Stelzner

G F

,

Hunsicker

P R

,

1988

Loftier frequency of mosaic mutants produced past N-ethyl-Due north-nitrosourea exposure of mouse zygotes

.

Proc. Natl. Acad. Sci. USA

85

:

9167

–

9170

.

Russell

W Fifty

,

1951

10-ray induced mutations in mice

.

Cold Leap Harbor Symp. Quant. Biol.

sixteen

:

327

–

336

.

Russell

W Fifty

,

Hunsicker

P R

,

Raymer

One thousand D

,

Steele

Yard H

,

Stelzner

K F

 et al. ,

1982

Dose-response curve for ethylnitrosourea-induced specific-locus mutations in mouse spermatogonia

.

Proc. Natl. Acad. Sci. USA

79

:

3589

–

3591

.

Savvides

South Northward

,

Boone

T

,

Andrew Karplus

P

,

2000

Flt3 ligand structure and unexpected commonalities of helical bundles and cystine knots

.

Nat. Struct. Biol.

7

:

486

–

491

.

Schumacher

A

,

Faust

C

,

Magnuson

T

,

1996

Positional cloning of a global regulator of anterior-posterior patterning in mice

.

Nature

384

:

648

.

Senapathy

P

,

Shapiro

M B

,

Harris

N L

,

1990

Splice junctions, branch point sites, and exons: sequence statistics, identification, and applications to genome project

.

Methods Enzymol.

183

:

252

–

278

.

Tajima

Y

,

Huang

E J

,

Vosseller

K

,

Ono

Chiliad

,

Moore

One thousand A

 et al. ,

1998

Function of dimerization of the membrane-associated growth factor kit ligand in juxtacrine signaling: the Sl17H mutation affects dimerization and stability-phenotypes in hematopoiesis

.

J. Exp. Med.

187

:

1451

–

1461

.

Wehrle-Haller

B

,

Imhof

B A

,

2001

Stalk cell factor presentation to c-Kit. Identification of a basolateral targeting domain

.

J. Biol. Chem.

276

:

12667

–

12674

.

Valentine

C R

,

1998

The association of nonsense codons with exon skipping

.

Mutat. Res.

411

:

87

–

117

.

Zhang

Z

,

Zhang

R

,

Joachimiak

A

,

Schlessinger

J

,

Kong

10 P

,

2000

Crystal construction of homo stem cell factor: implication for stalk jail cell factor receptor dimerization and activation

.

Proc. Natl. Acad. Sci. USA

97

:

7732

–

7737

.

Writer notes

i

Nowadays address: Horizon Molecular Medicine, Norcross, GA 30071.

2

Nowadays address: University Plan in Genetics, Duke University, Durham, NC 27710.

© Genetics 2002