An Allelic Series of Mutations in the Kit ligand Gene of Mice. I. Identification of Point Mutations in Seven Ethylnitrosourea-Induced Kitl^Steel Alleles

Corresponding author: M. A. Bedell, B416 Life Sciences, University of Georgia, Athens, GA 30602-7223., bedell{at}arches.uga.edu (E-mail)

Communicating editor: C. KOZAK

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

An allelic series of mutations is an extremely valuable genetic resource for understanding gene function. Here we describe eight mutant alleles at the Steel (Sl) locus of mice that were induced with N-ethyl-N-nitrosourea (ENU). The product of the Sl locus is Kit ligand (or Kitl; also known as mast cell growth factor, stem cell factor, and Steel factor), 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 Kitl^Sl 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 point mutations in Kitl. Interestingly, each of the five missense mutations affects residues that are within, or very near, conserved -helical domains of Kitl. These ENU-induced mutants should provide important information on structural requirements for function of Kitl and other helical cytokines.

THE ligand for Kit, a type 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 BESMER et al. 1993 ; LEV et al. 1994 ). Although sequence similarities have allowed assignment of Kit to the platelet-derived growth factor receptor, ß-polypeptide (Pdgfrb) family of RTKs, Kitl does not have significant sequence similarities to any 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 basis of limited sequence similarities (BAZAN 1991 ; HANNUM et al. 1994 ). Recent crystallographic studies have confirmed that Kitl (JIANG et al. 2000 ; ZHANG et al. 2000 ), Csf1 (PANDIT et al. 1992 ), and Flt3l (SAVVIDES et al. 2000 ) share a common structure and are members of the short-chain 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, 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 suggest that the Kitl/Csf1/Flt3l group shares more functional properties with the structurally different Pdgf/Vegf group than with the structurally more similar helical cytokine group (JIANG et al. 2000 ; SAVVIDES et 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 (FLANAGAN et al. 1991 ; HUANG et al. 1992 ). Alternative splicing produces two Kitl mRNAs (see below) that differ by the presence or absence of exon 6, which contains the primary site for proteolytic cleavage. The primary translation products of both Kitl mRNAs contain a 25-amino-acid (aa) signal sequence at the N terminus that is not found in the mature forms of the proteins (ANDERSON et al. 1990 ; MARTIN et al. 1990 ). The Kitl mRNA containing exon 6 [(+) E6, also called KL-1 (HUANG et al. 1992 )] encodes a 248-aa transmembrane precursor that produces a soluble isoform (S-Kitl) of 165 aa when processed at the primary cleavage site. The Kitl mRNA lacking exon 6 [(-) E6, also called KL-2 (HUANG et al. 1992 )] encodes a 220-aa transmembrane protein that lacks the primary cleavage site and is predominantly membrane bound (MB-Kitl). In the absence of the primary cleavage site, a secondary cleavage site in exon 7 of mouse Kitl is utilized, causing release of a second S-Kitl isoform (MAJUMDAR et al. 1994 ). Both S-Kitl and MB-Kitl form noncovalently linked dimers (ARAKAWA et al. 1991 ; HSU et al. 1997 ; TAJIMA et al. 1998 ) and are heavily glycosylated at both N- and O-linked sites (LU et al. 1991 , LU et al. 1992 ; HUANG et al. 1992 ). Although in vitro assays have demonstrated that S-Kitl and MB-Kitl are biologically active, MB-Kitl promotes a more persistent activation of Kit than does S-Kitl (MIYAZAWA et al. 1995 ).

Despite great interest in the biological activities of Kitl, relatively little is known of its sequence requirements for function. Only three studies of Kitl structure-function relationships in cultured cells have been reported (NISHIKAWA et al. 1992 ; LANGLEY et al. 1994 ; MATOUS et al. 1996 ). NISHIKAWA et al. 1992 constructed a series of C-terminal deletions and showed that hematopoietic progenitor cell growth in vitro requires only the first 142 aa of S-Kitl while minimal growth was observed with 133 aa of S-Kitl (NISHIKAWA et al. 1992 ). The importance of three of the four -helical domains to Kitl function was suggested by analysis of mouse-human chimeras of S-Kitl (MATOUS et al. 1996 ). These authors showed further that substitution of 4 aa in the fourth helical domain of S-Kitl abolished Kit binding and biological activity in vitro. While these studies have provided useful information on structural requirements for Kitl function 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 gene function. In mice, Kitl is encoded by the Sl locus and there are at least 80 different Kitl^Sl mutant alleles (MOUSE GENOME DATABASE 2002; MUTANT MOUSE DATABASE 2002). Previous studies have identified diverse molecular alterations in a number of Kitl^Sl mutant alleles. The most common type of Kitl^Sl mutation removes the entire Kitl coding region and ranges in size from 120-kb deletions to very large, cytogenetically visible deletions (CATTANACH et al. 1993 ; BEDELL et al. 1996A ). Other Kitl^Sl alleles (Kitl^Sl-pan and Kitl^Sl-con) contain chromosomal rearrangements that exert tissue-specific effects on Kitl mRNA expression, but leave intact the Kitl coding region (HUANG et al. 1993 ; BEDELL et al. 1995 ). Previous studies have also identified intragenic mutations that affect the Kitl coding region in five Kitl^Sl alleles. The Kitl^Sl-d allele contains an intragenic deletion and potentially encodes a nearly normal S-Kitl but completely lacks MB-Kitl (BRANNAN et al. 1991 ; FLANAGAN et al. 1991 ). The Kitl^Sl-17H allele contains a point mutation that affects splicing such that a MB-Kitl with an abnormal cytoplasmic domain is encoded but the mutation is not expected to affect production of S-Kitl (BRANNAN et al. 1992 ). Interestingly, recent evidence suggests that Kitl^Sl-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 Kitl^Sl-1Neu mutant, the 25-aa signal sequence and an additional 26 aa of the Kitl N terminus are missing and in the Kitl^Sl-2Neu mutant only 96 aa of the normal 165 aa of mature S-Kitl, plus two additional aa, are present (GRAW et al. 1996 ). In the Kitl^Sl-3Neu allele, a missense mutation has been identified that causes substitution of serine for asparagine at position 97 of the mature S-Kitl and MB-Kitl (GRAW et al. 1997 ). Although these five Kitl^Sl mutants have provided important information about Kitl function in vivo, a thorough understanding of this signaling molecule would be facilitated by the availability of a larger collection of intragenic Kitl^Sl alleles.

In this article we describe eight ethylnitrosourea (ENU)-induced Kitl^Sl mutant alleles, of which seven are shown to contain point mutations in the Kitl gene. While two of these point mutations are predicted to encode proteins with C-terminal deletions, five others encode missense substitutions that are located within, or very near, conserved -helical domains of Kitl. In the accompanying article (RAJARAMAN et al. 2002 , this issue) we describe the effects of these mutations on survival, pigmentation, and peripheral blood cells of mice. These mutations should help provide more information about specific residues that are required for Kitl function, as well as about function of structurally related cytokines.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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 (RUSSELL et al. 1982 ). All of the Kitl^Sl mutations except Kitl^Sl-39R were generated in stem-cell spermatogonia by mutagenesis of (101/Rl x C3H/Rl)F₁ or (C3H/Rl x 101/Rl)F₁ male mice, followed by mating of mutagenized mice to T-strain females (RUSSELL 1951 ). Kitl^Sl-39R was generated in zygotes by mating (B10/Rl x C3H/Rl)F₁ females to T-strain males, followed by ENU treatment of impregnated females (RUSSELL et al. 1988 ). The T-strain mice used for specific locus tests were homozygous for seven recessive alleles (a, Tyrp1^b, Tyr^c-ch, Myo5a^d, Bmp5^se, p, and Ednrb^s). Thus, new recessive mutations at these loci would be revealed among first-generation offspring of crosses between mutagenized, or control, (101/F₁ x C3H/Rl)F₁ 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 Kitl^Sl-12R, a known homozygous viable translocation, [T(10D;18D)12Rl], that affects the Kitl locus (CACHEIRO and RUSSELL 1975 ). Each of the Kitl^Sl alleles used in the present 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 by backcrossing heterozygous mice to inbred C3H/HeNCR mice. Kitl^Sl-gb mice, which carry an 120-kb deletion that removes the entire Kitl coding region as well as upstream sequences (BEDELL et al. 1996A ), were also maintained on a C3H/HEN CR background and were used in this study.

Total RNA isolation and RT-PCR sequencing analysis:
Total RNA was prepared from whole embryos or from kidneys and lungs of newborn mice using RNAzol (Tel-Test, Friendswood, TX) according to the manufacturer's instructions. Total RNA was used as a template for cDNA synthesis using reverse 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 (BEDELL et al. 1996B ). The sequence of the forward primer was 5'-CTATCTGCAGCCGCTGCTGG-3' and the sequence of the reverse primer was 5'-CTGTTACCAGCCACTGTGCG-3'. The (+) E6 and (-) E6 RT-PCR products (see Fig 1), which are 970 and 886 bp, respectively, were purified together using Wizard PCR Preps DNA 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 first set corresponds to sequences nested within the RT-PCR primers and provides sequence for both (+) E6 and (-) E6 RT-PCR templates, while the second set corresponds to sequences located within exon 6 and is therefore specific for (+) E6 templates. Nucleotide sequences were aligned and compared using Sequencher software (Gene Codes, Ann Arbor, MI).

View larger version (29K): In this window In a new window Download PPT slide   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.

Cloning of Kitl cDNAs:
The Kitl coding region of each mutant allele was amplified using primers analogous to those described above except that they contained restriction sites for the enzymes BamHI and PstI. PCR was performed using High Fidelity polymerase (Boehringer Mannheim, Indianapolis). The resulting PCR products were purified as described above, digested with BamHI and PstI, and cloned into BamHI and PstI sites of Bluescript plasmid (Stratagene, La Jolla, CA). For each mutant allele, two independent clones of the (+) E6 coding region and the (-) E6 coding region were isolated and sequenced using an automated sequencer.

Northern blot analysis:
Poly(A)+ RNA was prepared from the kidney and lung tissues of wild-type and homozygous mutant newborn mice using the Micro-Fast Track 2.0 kit (Invitrogen, Carlsbad, CA). For Northern blot analysis, the RNAs were electrophoresed through a 1% agarose/7% 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 SCF4.1 (Kitl) plasmid (ANDERSON et al. 1990 ) as template and reagents from the DIG RNA labeling kit (Boehringer Mannheim). Hybridization, washes, and detection were done according to the manufacturer's instructions. After stripping, the same blots were reprobed with a DIG-labeled antisense actin RNA to confirm uniform loading of sample RNAs.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Molecular genetic analysis:
All of the new Kitl^Sl mutant alleles in this study were generated following ENU treatment of F₁ mice. Although point mutations are the most common type of sequence alterations observed in germline mutations of mice following ENU treatment (NOVEROSKE et al. 2000 ), other types of genomic alterations are possible. To examine the structural integrity of the Kitl gene in each of the mutant alleles, Southern blot analysis of genomic DNA 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 3' untranslated region of Kitl, and a genomic fragment from the 5'-flanking region of Kitl (BEDELL et al. 1996B ). Together, these probes represent 40 kb of genomic DNA. The results indicate that there are no major structural alterations in the Kitl coding region and in flanking sequences in any of the ENU-induced alleles except Kitl^Sl-25R (data not shown). Analysis of the Kitl^Sl-25R allele revealed multiple restriction fragment length polymorphisms (RFLPs) that are consistent with intragenic rearrangements or deletions in the Kitl gene (data not shown).

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 Table 1). Of the seven mutations induced in (101/Rl x C3H/Rl)F₁ or (C3H/Rl x 101/Rl)F₁ mice, the Kitl^Sl-30R, Kitl^Sl-42R, Kitl^Sl-28R, and Kitl^Sl-25R mutations occurred on 101/Rl chromosomes while the Kitl^Sl-31R, Kitl^Sl-22R, and Kitl^Sl-36R mutations occurred on C3H/Rl chromosomes. The Kitl^Sl-39R mutation, which was generated from (B10/Rl x 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. Two RT-PCR products would be expected from wild-type tissues (see Fig 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. Because all coding exons are located in the amplified region, this amplification strategy should allow detection of any defects in mRNA splicing that affect the Kitl coding region. Following agarose gel electrophoresis, RT-PCR products of normal size were observed in samples from the Kitl^Sl-22R, Kitl^Sl-28R, Kitl^Sl-30R, Kitl^Sl-31R, Kitl^Sl-39R, and Kitl^Sl-42R tissues. However, from Kitl^Sl-36R tissues, an abnormally sized product of 645 bp was observed in addition to the expected 970- and 886-bp products in Kitl^Sl-36R (see Fig 1B and below). Multiple RT-PCR products of abnormal size, which are consistent with intragenic genomic alterations and abnormal splicing, were observed in Kitl^Sl-25R (not shown). Because of the complexity of these products and because the Kitl^Sl-25R allele behaves as a null allele (data not shown), the Kitl^Sl-25R RT-PCR products were not characterized further.

Nucleotide sequencing of RT-PCR products was performed for all alleles except Kitl^Sl-25R. RT-PCR products were generated using Taq polymerase and then purified and used directly as templates for nucleotide sequencing. Nucleotide sequence alterations observed with these Taq-generated products were confirmed by sequencing of at least two cloned RT-PCR products that were generated using High Fidelity polymerase. Using this strategy, we 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 by NOVEROSKE et al. 2000 ). These mutations are described in more detail (see below), and the Kitl sequences affected in each of these mutant alleles are illustrated in Fig 2.

View larger version (31K): In this window In a new window Download PPT slide   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 A, B, C, and D, -helical domains; boxes with horizontal lines, ß-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 Fig 1C and Fig 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 Fig 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 Fig 1B) is expected to produce a truncated protein of 96 aa + 25 out-of-frame aa.

Premature termination and exon skipping in Kitl^Sl-36R:
In the Kitl^Sl-36R allele, an abnormal-size RT-PCR product of 645 bp was observed in addition to the normal-size products of 970 and 886 bp (Fig 1B). Each of these three Kitl^Sl-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 5 was identified that creates a nonsense mutation in codon 147. Thus, one mutant product of only 146 aa of S-Kitl would be produced by premature termination of both of the (+) E6 and (-) E6 transcripts (Kitl^Sl-36R-A in Fig 2). In the 645-bp RT-PCR product, exon 4 was found to be spliced directly to exon 7 and is likely to result from abnormal splicing (skipping) of exon 5 as well as the normal splicing of exon 6 (Fig 1B). Sequencing of 10 cloned 645-bp RT-PCR products from Kitl^Sl-36R/Kitl^Sl-36R kidneys revealed that all 10 utilized the normal exon 4-exon 7 splice junctions (not shown). As the result of this exon skipping, sequences downstream to the exon 4-exon 7 splicing junction are out of frame. Thus, a second Kitl^Sl-36R isoform may be produced by exon skipping and is predicted to encode the first 96 aa of S-Kitl with 25 C-terminal aa out of frame (Kitl^Sl-36R-B in Fig 2).

A splicing defect in Kitl^Sl-42R:
Sequencing of the Kitl^Sl-42R RT-PCR products revealed an 8-bp insertion at the junction of exon 4 and exon 5 that creates a termination codon after two codons (Fig 1C and Fig D). As a result, this allele is predicted to encode the first 96 aa of S-Kitl with 2 C-terminal aa out-of-frame (Fig 2) and differs from the Kitl^Sl-36R-B isoform in only the out-of-frame C-terminal residues. In the Kitl^Sl-42R RT-PCR product, the 8-bp insertion differs by only 1 nt from a similar 8-bp insertion reported in the Kitl^Sl-2Neu allele (GRAW et al. 1996 ). Because both insertions are located at the junction of exon 4 and exon 5, it was possible that the insertions result from a splicing defect. To examine this hypothesis, we determined the sequence of the splice donor site of intron 4 from wild-type and Kitl^Sl-42R alleles. Intron 4 of wild-type genomic DNA was amplified using oligonucleotide primers from exons 4 and 5. Nucleotide sequencing revealed that the first 8 nucleotide (nt) of this intron differ from the Kitl^Sl-42R insertion by only 1 nt (see Fig 1E). Sequencing of the corresponding region of PCR-amplified genomic DNA from Kitl^Sl-42R/Kitl^Sl-42R tissue revealed the presence of a T C substitution in the 5' splice donor site (see Fig 1E). A second 5' splice donor site (GT) is located just 9 nt 3' of the normally used 5' donor site and is unaffected by the Kitl^Sl-42R mutation (wild type, 5'-GTAACTTGGT-3'; mutant, 5'-GCAACTTGGT-3'). Thus, the Kitl^Sl-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 determine the efficiency of normal and abnormal splicing events, we performed RT-PCR on mRNA from fetal liver of wild-type and Kitl^Sl-42R/Kitl^Sl-42R embryos using primers that flank the 8-bp insertion. The results revealed that there were no RT-PCR products of normal size detectable in Kitl^Sl-42R/Kitl^Sl-42R tissue (not shown). Thus, the vast majority of the Kitl^Sl-42R mRNA is aberrantly spliced such that little or no wild-type mRNA is expressed.

Missense mutations in five Kitl^Sl alleles:
Each of the five missense mutations affects sequences that are within or near conserved -helical domains of Kitl (see Fig 2). To gain insight into the functional importance of residues affected by the Kitl^Sl missense mutations, we also examined the conservation of each affected residue in orthologous sequences. Accordingly, the residues affected in each mutant are superimposed on a multiple sequence alignment of the processed S-Kitl orthologs from nine mammals in Fig 3. In addition, Fig 3 illustrates some structural aspects of human Kitl (from JIANG et al. 2000 ).

View larger version (56K): In this window In a new window Download PPT slide   Figure 3. 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, -helical domains; shaded rectangles, ß-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.

The Kitl^Sl-30R mutation results from a T325G transversion that causes a L18R missense mutation in A. The leucine at position 18 is conserved in all mammalian orthologs and affects a residue buried in the human Kitl dimer (Fig 3). Furthermore, the side-chains of leucine residues are thought to form the hydrophobic inner core of -helices and may be important for helix packing. Thus, replacement of the positively charged arginine for the hydrophobic, nonpolar leucine is likely to have a significant effect on protein conformation.

The Kitl^Sl-31R mutation results from a C340T transition that causes a P23L missense mutation at the very end of A. Interestingly, the proline at position 23 in Kitl is conserved in all mammalian orthologs (Fig 3) and prolines flanked by polar residues are found frequently at the termini of -helices (GUNASEKARAN et al. 1998 ). Thus, the P23L replacement in Kitl may affect the termination of A. Furthermore, this substitution affects a residue at the human Kitl dimer interface (JIANG et al. 2000 ), suggesting that the Kitl^Sl-31R encoded protein may not be able to dimerize efficiently.

The Kitl^Sl-22R mutation results from a T433C transition that causes a L54P missense mutation in B. 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 alteration identified in RT-PCR products (not shown). Unlike residues affected by the other Kitl missense mutations, the leucine at position 54 is not conserved but is a valine, another hydrophobic, nonpolar amino acid, in seven of the nine mammalian orthologs (Fig 3). However, prolines are generally incompatible with -helical domains and the presence of the L54P replacement within B, like the L18R replacement in A of the Kitl^S-30R mutant, is likely to have a significant effect on local tertiary structure.

The Kitl^Sl-28R mutation results from a T626A transversion that causes an I118N missense mutation in D. The isoleucine at position 118 is found in all nine mammalian orthologs (Fig 3). Substitution of the polar asparagine residue for the hydrophobic, buried isoleucine residue would be expected to have a major effect on local structure of that region.

The Kitl^Sl-39R mutation results from a C637T transition that causes a S122F missense mutation in D. This mutation abolishes a MboI restriction enzyme site (GATC) and creates a HinfI restriction site (GATTC). These alterations were confirmed using restriction enzyme digestion of RT-PCR products from wild-type and Kitl^Sl-39R tissues (not shown). The serine at position 122 is conserved in all nine mammalian orthologs (Fig 3) and is a part of a recognition sequence for N-linked glycosylation, i.e., Asn-X-Ser/Thr. Consequently, the S122F substitution would disrupt one of four N-linked glycosylation sites identified in mouse Kitl (see Fig 2). Although in vitro studies with recombinant S-Kitl have indicated that glycosylation is not essential for biological activity (LANGLEY et al. 1994 ), N-linked glycans are known to facilitate protein folding and conformational maturation (HELENIUS 1994 ; O'CONNOR and IMPERIALI 1996 ). Thus, altered glycosylation of Kitl^Sl-39R-encoded Kitl may contribute to altered function in vivo.

Steady-state levels of mutant Kitl mRNAs:
To determine whether any of the mutations affect steady-state levels of Kitl mRNA, Northern blot analysis of tissues from mice homozygous for each mutation was performed. To ensure that the probe did not cross-hybridize to other mRNAs, a control lane contained RNA prepared from tissues of Kitl^Sl-gb/Kitl^Sl-gb mice, which are homozygous for a deletion that removes the Kitl coding region (BEDELL et al. 1996A ). RNAs from at least two mice homozygous for each mutant allele were analyzed in separate Northern blots and each blot contained lanes with RNA from Kitl⁺/Kitl⁺ and Kitl^Sl-gb/Kitl^Sl-gb mice. A representative Northern blot is shown in Fig 4. Although a small amount of variation was noted for samples of the same genotype on different blots, the combined results from all blots reveal that all mutant alleles except Kitl^Sl-gb express a Kitl transcript of similar size and abundance to that of the wild-type allele. Because these studies were performed on lung and kidney of newborn mice, we cannot exclude the possibility of tissue-specific or developmental-stage-specific effects on Kitl mRNA abundance. However, 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.

View larger version (101K): In this window In a new window Download PPT slide   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 ß-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+.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In this report we describe the molecular genetic alterations in eight Kitl^Sl mutant alleles that arose from ENU mutagenesis. One of these alleles (Kitl^Sl-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 Kitl^Sl alleles and were of five types: two A/T G/C transitions (Kitl^Sl-22R and Kitl^Sl-42R); two G/C A/T transitions (Kitl^Sl-31R and Kitl^Sl-39R); an A/T T/A transversion (Kitl^Sl-28R); an A/T C/G transversion (Kitl^Sl-30R); and a G/C T/A transversion (Kitl^Sl-36R). Such molecular diversity is somewhat surprising considering that 82% of the previously characterized ENU-induced germline mutations in the mouse were either A/T G/C transitions or A/T T/A transversions (NOVEROSKE et al. 2000 ). The only previously reported ENU-induced Kitl^Sl mutation was an A/T T/A transversion in the Kitl^Sl-17H allele (BRANNAN et al. 1992 ). Two aspects of the ENU-induced Kitl^Sl allelic series are noteworthy. First, the Kitl^Sl-36R is only the second G/C T/A transversion reported for an ENU-induced mouse germline mutation (NOVEROSKE et al. 2000 ). This type of mutation has been reported previously only in a Pax6^Sey mutant allele (HILL et al. 1991 ). Second, the G/C A/T transition in the Kitl^Sl-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 between the two types of cells. Together, our observations of Kitl^Sl alleles add to the notion that ENU causes predominantly point mutations in mice (MARKER et al. 1997 ; JUSTICE et al. 1999 ; NOVEROSKE et al. 2000 ).

The mutagenesis tests that generated the present mutations were designed to detect recessive mutations at seven defined loci and visible dominant mutations at other loci (RUSSELL 1951 ; RUSSELL et al. 1982 ). The new Kitl^Sl alleles were detected because they exhibited semidominant pigmentation phenotypes (described in RAJARAMAN et al. 2002 ). Including previously reported Kitl^Sl alleles (KURODA et al. 1988 ; COPELAND et al. 1990 ; BRANNAN et al. 1991 , BRANNAN et al. 1992 ; CATTANACH et al. 1993 ; BEDELL et al. 1995 , BEDELL et al. 1996A ; GRAW et al. 1996 , GRAW et al. 1997 ) and unpublished Kitl^Sl alleles (MOUSE GENOME DATABASE 2002; MUTANT MOUSE DATABASE 2002), this brings the total number of Kitl^Sl mutant alleles to at least 80. Such a large allelic series exists at only a handful of other loci in the mouse (see MOUSE GENOME DATABASE 2002; MUTANT MOUSE DATABASE 2002 and review by DAVIS and JUSTICE 1998 ), notably loci included in the specific locus test (a, Tyr1^b, Tyr^c, Myo5a^d, Bmp5^se, p, and Ednrb^s), as well as Mitf^Mi and Kit^W. In the Kitl^Sl allelic series, the variation in phenotypic severity and diversity of molecular defects is quite remarkable. Some Kitl^Sl alleles contain deletions that completely remove the Kitl gene, as well as closely linked genes (CATTANACH et al. 1993 ; BEDELL et 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 hand, are very valuable because they may allow phenotypic analyses at developmental stages later than those reached by null mutants and may reveal more subtle effects (SCHUMACHER et al. 1996 ). Previously characterized hypomorphic Kitl^Sl alleles include two that affect expression of Kitl mRNA (Kitl^Sl-pan and Kitl^Sl-con, BEDELL et al. 1995 ), four that affect entire domains of the Kitl protein (Kitl^Sl-d, BRANNAN et al. 1991 and FLANAGAN et al. 1991 ; Kitl^Sl-17H, BRANNAN et al. 1992 ; Kitl^Sl-1Neu and Kitl^Sl-2Neu, GRAW et al. 1996 ), and one missense mutation in Kitl (Kitl^Sl-3Neu, GRAW et al. 1997 ). While two of the Kitl^Sl alleles in the present report also affect entire domains of Kitl because of premature termination and aberrant mRNA splicing (Kitl^Sl-36R and Kitl^Sl-42R), five alleles were found to contain missense mutations (Kitl^Sl-30R, Kitl^Sl-31R, Kitl^Sl-22R, Kitl^Sl-28R, and Kitl^Sl-39R). These alleles should provide important information on critical residues and domains that are required for Kitl function. In the accompanying article (RAJARAMAN et al. 2002 ) we provide evidence that five of these new alleles (Kitl^Sl-30R, Kitl^Sl-31R, Kitl^Sl-22R, Kitl^Sl-28R, and Kitl^S-42R) have little or no functional activity for mouse survival or development of peripheral blood cells while two of these alleles are hypomorphic (Kitl^Sl-36R and Kitl^Sl-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 (JUSTICE et al. 1999 ). Consistent with this observation, we describe two mutations (Kitl^Sl-42R and Kitl^Sl-36R) that affect Kitl mRNA splicing. However, the mechanisms by which the aberrant splicing occurs are distinctly different in these mutants. The Kitl^Sl-42R allele contains a point mutation in the normal splice donor site of intron 4 (Fig 1D and Fig E) 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 there is substantial evidence that these nucleotides are essential to correct mRNA splicing (SENAPATHY et al. 1990 ). In contrast, nucleotide sequence analysis of RT-PCR products from the Kitl^Sl-36R allele predicts two molecular defects: the creation of a nonsense mutation in exon 5, which is present in both the (+) E6 and (-) E6 alternative transcripts, and skipping of exon 5, which generates the (-) E5, (-) E6 cDNA (Fig 1B). Since the first report in 1993 (DIETZ et 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 30 bp of exon-intron boundaries and usually involve substitution to a thymidine residue (VALENTINE 1998 ). Consistent with this, the nonsense mutation in Kitl^Sl-36R is created by a G T transversion that occurs within a GAGAAAG sequence (where the underlined G is converted to T in the mutant) that immediately precedes that boundary between exon 5 and intron 5.

Although truncation mutants are useful for understanding the role(s) of different protein domains, missense mutations are most useful for identifying critical residues that affect structure and function. At present, we do not know the mechanism by which the Kitl^Sl missense mutations affect Kitl function. Because our studies suggest that the steady-state level of Kitl mRNA expressed by each allele is normal (Fig 4), it is likely that some aspect of Kitl structure or function is affected. Since all of the Kitl missense mutations are within or very near -helical domains, it is very likely that loss of function 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 Kitl^Sl-39R mutant, as this point mutation affects a site for N-linked glycosylation at Asn 120 (LU et al. 1991 , LU et al. 1992 ). The last possibility is that the mutant Kitl may not interact properly with Kit and may exhibit either quantitative or qualitative differences in binding to and activating the receptor. Further studies that examine each of the above aspects of Kitl processing, localization, and binding to Kit by each of the Kitl^Sl mutants will be necessary.

	FOOTNOTES

¹ Present address: Horizon Molecular Medicine, Norcross, GA 30071.
² Present address: University Program in Genetics, Duke University, Durham, NC 27710.

	ACKNOWLEDGMENTS

We thank Drs. Neal Copeland and Nancy Jenkins for their support during the early stages of these experiments and for providing resources for embryo rederivation and Keycharianne Gómez for technical assistance. We also thank Drs. Eugene Rinchik and David Williams and two anonymous reviewers for their comments on the manuscript. The mutants were generated in research jointly sponsored by the Office of Biological and Environmental Research, USDOE, at the Oak Ridge National Laboratory, managed by UT-Batelle, LLC, under contract DE-AC05-00OR22725, and by the National Institute of Environmental Health Sciences under interagency agreement no. Y1-ES-8048/0524-I119-A1. The material in this manuscript is based on work supported by the National Science Foundation under grant no. IBN-9728428 and the University of Georgia Research Foundation.

Manuscript received February 27, 2002; Accepted for publication June 26, 2002.

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