The role of a single formin isoform in the limb and renal phenotypes of limb deformity.

BACKGROUND: Mutations of the murine limb deformity (ld) locus are responsible for a pleiotropic phenotype of completely penetrant limb malformations and incompletely penetrant renal agenesis and/or dysgenesis. The ld locus encodes a complex family of mRNA and protein isoforms. MATERIALS AND METHODS: To examine the role of one of the more prominent of these isoforms, isoform IV, we specifically eliminated it by gene targeting. RESULTS: Unlike other mutant ld mice, homozygous mice bearing this isoform IV disruption display incompletely penetrant renal agenesis, but have perfectly normal limbs. Whole mount in situ hybridization demonstrated that this targeted disruption was specific for isoform IV and did not interfere with the expression of other ld isoforms. The isoform IV-disrupted allele of ld does not complement the renal agenesis phenotype of other ld alleles, in a manner consistent with its penetrance, and like the isoform IV-deficient mice, these compound heterozygotes have normal limbs. Sequence analysis of formin isoform IV in other ld mutant alleles did not detect any amino acid changes relative to the strain of origin of the mutant allele. CONCLUSIONS: Thus, the disruption of isoform IV is sufficient for the renal agenesis phenotype, but not the limb phenotype of ld mutant mice. Structural mutations in this isoform are only one of several genetic mechanisms leading to the renal phenotype, since amino acid changes in this isoform were not detected. These results demonstrate that this gene is limb deformity, and that variable isoform expression may play a role in generating the pleiotropic ld phenotype.     

The Antimorphic Nature of the T(c) Allele at the Mouse T Locus

The T locus on mouse chromosome 17 is haploid-insufficient: deletion/+ heterozygous mice have a short tail. One exceptional allele, Tc, produces a tailless phenotype in heterozygous mice. Thus, Tc has a more severe phenotype than that of a deletion allele, suggesting either that Tc is further deleted for a neighboring locus, resulting in the additional phenotype, or that Tc is a gain-of-function mutation. We have shown that Tc is not deleted for the D17Leh119 and D17RP17 loci flanking T, which are deleted in some T alleles. Thus, the severity of the Tc phenotype is not due to the deletion of an adjacent locus. We have also examined the genetic nature of the Tc allele by placing it in trans with a T-locus duplication, twLub2, which has previously been independently confirmed at the molecular level to have a duplication in the chromosomal region including the T locus. We have shown that Tc is partially complemented by twLub2, unlike a null allele (deletion) which was previously shown to be fully complemented by twLub2. These results indicate that Tc behaves genetically as an antimorph, exerting its effect by antagonizing the function of a wild-type allele at the T locus. The apparent correlation between the gene dosage at the T locus and the length of the body axis is discussed.     

Molecular analysis of two mouse dilute locus deletion mutations: spontaneous dilute lethal20J and radiation-induced dilute prenatal lethal Aa2 alleles.

The dilute (d) coat color locus of mouse chromosome 9 has been identified by more than 200 spontaneous and mutagen-induced recessive mutations. With the advent of molecular probes for this locus, the molecular lesion associated with different dilute alleles can be recognized and precisely defined. In this study, two dilute mutations, dilute-lethal20J (dl20J) and dilute prenatal lethal Aa2, have been examined. Using a dilute locus genomic probe in Southern blot analysis, we detected unique restriction fragments in dl20J and Aa2 DNA. Subsequent analysis of these fragments showed that they represented deletion breakpoint fusion fragments. DNA sequence analysis of each mutation-associated deletion breakpoint fusion fragment suggests that both genomic deletions were generated by nonhomologous recombination events. The spontaneous dl20J mutation is caused by an interstitial deletion that removes a single coding exon of the dilute gene. The correlation between this discrete deletion and the expression of all dilute-associated phenotypes in dl20J homozygotes defines the dl20J mutation as a functional null allele of the dilute gene. The radiation-induced Aa2 allele is a multilocus deletion that, by complementation analysis, affects both the dilute locus and the proximal prenatal lethal-3 (pl-3) functional unit. Molecular analysis of the Aa2 deletion breakpoint fusion fragment has provided access to a previously undefined gene proximal to d. Initial characterization of this new gene suggests that it may represent the genetically defined pl-3 functional unit.     

Physical and Genetic Characterization of a 75-Kilobase Deletion Associated with A(l), a Recessive Lethal Allele at the Mouse Agouti Locus

The agouti locus (A) of the mouse determines the timing and type of pigment deposition in the growing hair bulb, and several alleles at this locus are lethal when homozygous. Apparent instances of intragenic recombination and complementation between different recessive lethal alleles have suggested that the locus has a complex structure. We have begun to investigate the molecular basis of agouti gene action and recessive lethality by using a series of genetically linked DNA probes and pulsed field gel electrophoresis to detect structural alterations in radiation-induced agouti mutations. Hybridization probes from the Src and Emv-15 loci do not reveal molecular alterations in DNA corresponding to the ae, ax, and al alleles, but a probe from the parotid secretory protein gene (Psp) detects a 75-kilobase (kb) deletion in DNA containing the non-agouti lethal allele (al). The deletion is defined by a 75-kb reduction in the size of BssHII, NotI, NruI and SacII high molecular weight restriction fragments detected with the Psp probe and is located between 25 kb and 575 kb from Psp coding sequences. Because the genetic distance between A and Emv-15 is much less than A and Psp, there may be a preferred site of recombination close to Psp, or suppression of recombination between A and Emv-15. The al deletion has allowed us to determine the genotype of mice heterozygous for different recessive lethal alleles. We find that three different recessive lethal complementation groups are present at the agouti locus, two of which are contained within the al deletion.     

Genetic variants of the Bombyx mori silkworm encoding sericin proteins of different lengths

A variant sericin polypeptide originally found by acid gel electrophoresis in the Nd-s mutant strain of the silkworm, Bombyx mori, has been analyzed genetically. The vriant polypeptide (called S-2^v) is encoded by a gene which behaves as a codominant allele of the gene encoding the standard S-2 sericin polypeptide. Linkage analysis locates these alleles at 0.0 map unit on chromosome 11. SDS-polyacrylamide gel electrophoresis shows that the molecular weight of the S-2^v variant polypeptide is lower by approximately 62,500 than that of the S-2 polypeptide. Amino acid analysis indicates that the two sericin polypeptides have similar compositions. These results are consistent with the idea that the variant allele arose by deletion within the S-2 coding sequence in the Src-2 gene locus as the result of unequal recombination.     

Evidence for WT1 as a Wilms tumor (WT) gene: intragenic germinal deletion in bilateral WT.

The inactivation of two alleles at a locus on the short arm of chromosome 11 (band 11p13) has been suggested to be critical steps in the development of Wilms tumor (WT), a childhood kidney tumor. Two similar candidate WT cDNA clones (WT33 and LK15) have recently been identified on the basis of both their expression in fetal kidney and their location within the smallest region of overlap of somatic 11p13 deletions in some tumors. These homozygous deletions, however, are large and potentially affect more than one gene. Using a cDNA probe to the candidate gene, we have analyzed DNA from both normal and tumor tissue from WT patients, in an effort to detect rearrangements at this locus. We report here a patient with bilateral WT who is heterozygous for a small (less than 11 kb) germinal deletion within this candidate gene. DNA from both tumors is homozygous for this intragenic deletion allele, which, by RNA-PRC sequence analysis, is predicted to encode a protein truncated by 180 amino acids. These data support the identification of this locus as an 11p13 WT gene (WT1) and provide direct molecular data supporting the two-hit mutational model for WT.     

A Genetic Characterization of the Nadc Gene of Salmonella Typhimurium

The nadC gene of Salmonella encodes the pyridine biosynthetic enzyme PRPP-quinolinate phosphoribosyltransferase. Using a combination of genetic techniques, a deletion map for the Salmonella nadC gene has been generated which includes over 100 point mutants and 18 deletion intervals. The nadC alleles obtained by hydroxylamine mutagenesis include those suppressed by either amber, ochre, or UGA nonsense suppressors as well as alleles suppressed by the missense suppressor, sumA. Deletions were obtained by three separate protocols including spontaneous selection for loss of the nearby aroP gene, recombination between aroP::MudA and nadC::MudA insertion alleles, and selection for spontaneous loss of tetracycline resistance in a nearby guaC::Tn10dTc insertion mutant allele. The nadC mutants comprise one complementation group and the nadC+ allele is dominant to simple, nadC auxotrophic mutant alleles. Intragenic complementation of two nadC alleles, nadC493 and nadC494, mapping to deletion intervals 17 and 18, respectively, suggests that nadC encodes a multimeric enzyme. Both nadC and the nearby aroP locus are transcribed counterclockwise on the standard genetic map of Salmonella, in opposite orientation to the direction of chromosome replication.     

A small deletion hotspot in the type II keratin gene mK6irs1/Krt2-6g on mouse chromosome 15, a candidate for causing the wavy hair of the caracul (Ca) mutation

A new mutation has arisen in a colony of mice transgenic for human alpha-galactosidase. The mutation is independent of the transgenic insertion, autosomal dominant, and morphologically very similar to the classical wavy coat mutation, caracul (Ca), on chromosome 15. Therefore, we designated this locus the caracul Rinshoken (Ca(Rin)). Applying a positional cloning approach, we identified the mK6irs1/Krt2-6g gene as a strong candidate for Ca(Rin) because among five Ca alleles examined mutations always occurred in the highly conserved positions of the alpha-helical rod domain (1A and 2B subdomain) of this putative gene product. The most striking finding is that four independently discovered alleles, the three preexistent alleles Ca(J), Ca(9J), Ca(10J), and our allele Ca(Rin), all share one identical amino acid deletion (N 140 del) and the fifth, Ca(medJ), has an amino acid substitution (A 431 D). These findings indicate that a mutation hotspot exists in the Ca locus. Additionally, we describe a Ca mutant allele induced by ENU mutagenesis, which also possesses an amino acid substitution (L 424 W) in the mK6irs1/Krt2-6g gene. The identification of the Ca candidate gene enables us to further define the nature of the genetic pathway required for hair formation and provides an important new candidate that may be implicated in human hair and skin diseases.     

Mutations in the melanocortin 1 receptor (MC1R) gene are associated with coat colours in the domestic rabbit (Oryctolagus cuniculus)

We sequenced almost the complete coding region of the MC1R gene in several domestic rabbits (Oryctolagus cuniculus) and identified four alleles: two wild-type alleles differing by two synonymous single nucleotide polymorphisms (c.333A>G;c.555T>C), one allele with a 30-nucleotide in-frame deletion (c.304_333del30) and one allele with a 6-nucleotide in-frame deletion (c.280_285del6). A polymerase chain reaction-based protocol was used to distinguish the wild-type alleles from the other two alleles in 263 rabbits belonging to 37 breeds or strains. All red/fawn/yellow rabbits were homozygous for the c.304_333del30 allele. This allele represents the recessive e allele at the extension locus identified through pioneering genetic studies in this species. All Californian, Checkered, Giant White and New Zealand White rabbits were homozygous for allele c.280_285del6, which was also observed in the heterozygous condition in a few other breeds. Black coat colour is part of the standard colour in Californian and Checkered breeds, in contrast to the two albino breeds, Giant White and New Zealand White. Following the nomenclature established for the rabbit extension locus, the c.280_285del6 allele, which is dominant over c.304_333del30, may be allele E(D) or allele E(S).     

Independent origins of self-compatibility in Arabidopsis thaliana

The evolution from outcrossing based on self-incompatibility (SI) to a selfing system is one of the most prevalent transitions in flowering plants. It has been suggested that the loss of SI in Arabidopsis thaliana is associated with pseudogene formation at the SCR male component of the S locus. Recent work, however, suggests that alternative alleles with large deletions at the S locus are also present and may be responsible for the evolution of self-compatibility in this species. We demonstrate that most of these deletion alleles are evolutionarily derived from an S haplotype (haplogroups A) that already possessed the SCR pseudogene. This haplotype and its deletion variants are nearly fixed in Europe. Together with previous transgenic data, these results suggest that the pseudogenization of PsiSCR1 gene changed the SI phenotype in the majority of A. thaliana accessions, and was a critical step in the evolution of selfing in this species. Two other haplogroups (B and C) were also identified, the former of which contains a novel and possibly functional SCR allele. In contrast to haplogroups A, these two haplogroups are found primarily in Africa and Asia. These results suggest that self-compatibility, which appears to be fixed in this species, arose multiple times with different genetic bases, and indicates that a species-specific trait is associated with parallel evolution at the molecular level.     

Discovery of a new fragrance allele and the development of functional markers for the breeding of fragrant rice varieties

The recessive fgr gene on chromosome 8 is associated with rice fragrance. It has been reported that this gene is a non-functional badh2 allele and that the functional Badh2 allele encoding putative betaine aldehyde dehydrogenase (BADH2) could render rice non-fragrant. Here we report the discovery of a new badh2 allele and the development of functional markers for the badh2 locus. A total of 24 fragrant and ten non-fragrant rice varieties were studied and sequenced for their Badh2/badh2 loci. Of the 24 fragrant rice varieties, 12 were found to have the known badh2 allele (badh2-E7), which has an 8-bp deletion and three single nucleotide polymorphisms (SNPs) in exon 7; the others had a novel null badh2 allele (badh2-E2), which has a sequence identical to that of the Badh2 allele in exon 7, but with a 7-bp deletion in exon 2. Both null badh2 alleles are responsible for rice fragrance. Based on sequence divergence amongst the functional Badh2 and two null badh2 alleles, we developed functional markers which can be easily used to distinguish non-fragrant from fragrant rice and to differentiate between two kinds of fragrant rice. These functional markers will find their usefulness in breeding for fragrant rice varieties via marker-assisted selection. Weiwei Shi and Yi Yang contributed equally to this work.     

Molecular evidence for compound heterozygosity in hereditary fructose intolerance.

Hereditary fructose intolerance (HFI) is an inborn error of metabolism, inherited as an autosomal recessive disorder and caused by a decrease in the activity of fructose-1-phosphate aldolase (aldolase B) in affected individuals. Investigation of the molecular basis of HFI is reported here by the identification of two molecular lesions in the aldolase B gene of the HFI individual. Using polymerase chain reaction to specifically amplify exons at this locus and T7 polymerase for the sequence determination of these double-stranded fragments, we show the mutational heterogeneity of the proband. One allele, previously indicated by restriction analysis, was confirmed as A149P (Ala 149 to Pro in exon 5). The other allele was identified as a 4-bp deletion found in exon 4, a deletion which causes a frameshift at codon 118, resulting in a truncated protein of 132 amino acids. Segregation of these mutant alleles in the proband's family was shown by using allele-specific oligodeoxynucleotides to probe blots of amplified DNA. The techniques employed here represent a rapid and efficient method for detection of other mutations in families with this disease. In addition, the ability to detect mutant alleles by allele-specific hybridization offers a new method for definitive diagnosis, a method which avoids a fructose loading or liver-biopsy examination.     

Frequency and rearrangements of alleles of proto-oncogene c-Ha-ras-1 and their association with development of various malignant tumors in man

The c-Ha-ras-1 locus in 84 cancer patients was examined for allelic restriction fragment length's polymorphism, as well as for distribution of four common c-Ha-ras-1 alleles (a1, a2, a3 and a4) in lung, ovarian and thyroid cancer patients. In approximately half (8 out of 15) lung and ovarian carcinomas possessing a4 allele, alterations in the Ha-ras locus (deletion, amplification and change in allele length) were detected, as compared to 2 cases of rearrangements out of 40 tumors lacking the a4 allele. An increased a4 allele frequency was found in individuals with lung and ovarian carcinomas, as compared to both controls--summarized literature data, and thyroid cancer patients. On the other hand, homozygosity for the a2 locus, resulting from deletion in another allele, and increased a2 allele frequency in thyroid cancer patients were observed. Thus, a4 and a2 alleles of the c-Ha-ras-1 may perhaps be viewed as genetic markers of predisposition to lung, ovarian and thyroid cancer, respectively, in combination with other clinical parameters.     

ENU-induced allele of brachyury (Tkt1) exhibits a developmental lethal phenotype similar to the original brachyury (T) mutation

New alleles of brachyury (Tkt1, Tkt4) were induced in the mouse complete tw5 haplotype by ethylnitrosourea (ENU). Like the original brachyury (T) mutation, the new alleles cause a short-tailed phenotype in heterozygotes, and interact with the t complex tail interaction factor (tct) in trans to cause phenotypically tailless mice. Because ENU is mainly a point mutagen, it is important to determine that the new alleles are homozygous embryonic lethal mutations like the original T allele, and to characterize their embryonic lethal phenotype. Moreover, the Tkt1 mutation maps to an inverted position relative to quaking (qk) in t haplotypes as compared with its position on normal chromosome 17. The Tkt1 allele was separated from the resident tw5 lethal gene, tclw5, by recombination, allowing embryology studies to be performed. Embryological analyses show that the Tkt1 allele is nearly identical to the classic T allele. At 9 and 10 days of development, homozygous Tkt1/Tkt1 embryos are grossly abnormal with properties including 1) irregular, disorganized somite pairs, 2) a shortened posterior end of the embryo, 3) an irregular neural tube, and 4) an abnormal notochord. In addition, 10 day-old abnormal embryos have anterior limb buds that point dorsally rather than ventrally, and are smaller than normal littermates. We conclude that the Tkt1 mutation is a valuable allele for both mapping and molecular characterization of the brachyury locus.     

Recessive allelic variations of three microsatellite sites within the<Emphasis Type="Italic">O2</Emphasis> gene in maize

Recessive allelic variations were investigated at 3 microsatellite (SSR) sites within theO2 gene by using 14 inbredo2 lines and a wild-type line in maize. Among the 15 lines, allelic variations were observed at umc1066, phi057, and phi112 sites. Two alleles were found at the umc1066 site—a recessive allele with 2 perfect GCCAGA repeats and a dominant allele with 3 perfect repeats. Three alleles were found at the phi057 site—2 recessive alleles with 3 and 5 perfect GCC repeats, respectively, and another with 4 perfect repeats consistent with a dominant allele. At least 4 alleles exist at the phi112 site—among which 1 recessive allele has a 1-bp deletion, another has a 15-bp deletion, and other has no PCR products compared to the dominant allele; all the alleles have unchanged AG repeats. The phi057 site in exon 6 was identified to be a hypervariable region in the coding sequence of the02 gene, in addition to the 2 hypervariable regions in exon 1 previously reported. The primary mechanisms underlying the variations in repeat numbers and regions flanking the SSR within theO2 gene appear to be unequal crossing over and replication slippage. Furthermore, base substitution of SSR motif can create heteroalleles and modify the repeat number of SSR. The lysine content of kernel in theO2 ando2 lines correlates to a considerable extent with nucleotide variations at the umc1066, phi057, and phi112 sites. Our study suggests that it is best to use the 3 markers together in molecular marker-assisted selection for high-lysine maize materials.     

High-resolution mapping of the Gli3 deletion in the mouse extra-toesH mutant

Extra-toes is a semidominant mutation that affects the Gli3 gene and provokes limb and brain abnormalities. Among the different alleles of this mutation, Xt(H) is due to a deletion that has not yet been fully characterized. Using a PCR-based strategy, we undertook a high-resolution mapping of this deletion and confirmed that Xt(H) is a null allele of Gli3. We further designed a PCR test to identify unequivocally heterozygous and homozygous embryos from their wild-type littermates. Despite the length of the Xt(H) deletion, available data on the mouse genome indicate that no genes other than Gli3 are deleted in Xt(H) mutants. Thus, the Xt(H) mutation can be used as a model for studying the effects that absence of Gli3 function has during development.