Spontaneous splicing mutations at the dihydrofolate reductase locus in Chinese hamster ovary cells.

We isolated and characterized three spontaneous mutants of Chinese hamster ovary cells that were deficient in dihydrofolate reductase activity. All three mutants contained no detectable enzyme activity and produced dihydrofolate reductase mRNA species that were shorter than those of the wild type by about 120 bases. Six exons are normally represented in this mRNA; exon 5 was missing in all three mutant mRNAs. Nuclease S1 analysis of the three mutants indicated that during the processing of the mutant RNA, exon 4 was spliced to exon 6. The three mutant genes were cloned, and the regions around exons 4 and 5 were sequenced. In one mutant, the GT dinucleotide at the 5' end of intron 5 had changed to CT. In a second mutant, the first base in exon 5 had changed from G to T. In a revertant of this mutant, this base was further mutated to A, a return to a purine. Approximately 25% of the mRNA molecules in the revertant were spliced correctly to produce an enzyme with one presumed amino acid change. In the third mutant, the AG at the 3' end of intron 4 had changed to AA. A mutation that partially reversed the mutant phenotype had changed the dinucleotide at the 5' end of intron 4 from GT to AT. The splicing pattern in this revertant was consistent with the use of cryptic donor and acceptor splice sites close to the original sites to produce an mRNA with three base changes and a protein with two amino acid changes. These mutations argue against a scanning model for the selection of splice site pairs and suggest that only a single splice site need be inactivated to bring about efficient exon skipping (a regulatory mechanism for some genes). The fact that all three mutants analyzed exhibited exon 5 splicing mutations indicates that these splice sites are hot spots for spontaneous mutation.     

Isolation and characterization of sequence‐tagged microsatellite sites markers in chickpea (Cicer arietinum L.)

In this study we report the isolation of microsatellite sequences and their conversion to sequence‐tagged microsatellite sites (STMS) markers in chickpea (Cicer arietinum L.). Thirteen putative recombinants isolated from a chickpea genomic library were sequenced, and used to design 10 STMS primer pairs. These were utilized to analyse the genetic polymorphism in 15 C. arietinum varieties and two wild varieties, C. echinospermum and C. reticulatum. All the primer pairs amplified polymorphic loci ranging from four to seven alleles per locus. The observed heterozygosity ranged from 0 to 0.6667. Most of the STMS markers also amplified corresponding loci in the wild relatives suggesting conservation of these markers in the genus. Hence, these polymorphic markers will be useful for the evaluation of genetic diversity and molecular mapping in chickpea.     

Polymorphism of fecundity genes (FecB, FecX, and FecG) in the Indian Bonpala sheep

The present study was designed for screening polymorphism of known fecundity genes in prolific Indian Bonpala sheep. Employing tetra-primer amplification refractory mutation system PCR, 11-point mutations of BMP1B, BMP15, and GDF9 genes of 97 Bonpala ewes were genotyped. The FecB locus of the BMPR1B gene and two loci (G1 and G4) of GDF9 gene were found to be polymorphic. In FecB locus, three genotypes, namely, wild type (Fec++, 0.02), heterozygous (FecB+, 0.23), and mutant (FecBB, 0.75) were detected. At G1 locus of GDF9 gene, three genotypes, namely, wild type (GG, 0.89), heterozygous (GA, 0.10), and mutant (AA, 0.01) were detected. At G4 locus of GDF9 gene, three genotypes, namely, wild type (AA, 0.01), heterozygous (AG, 0.14), and mutant (GG, 0.85) were detected. Statistically no significant correlation of polymorphism of FecB, G1, and G4 loci and litter size was found in this breed. All five loci of BMP15 and three loci of GDF 9 genes were monomorphic. This study reports Bonpala sheep as the first sheep breed where concurrent polymorphism at three important loci (FecB, G1, and G4) of two different fecundity genes (BMPR1B and GDF9) has been found.     

Mutations in the Unc-52 Gene Responsible for Body Wall Muscle Defects in Adult Caenorhabditis Elegans Are Located in Alternatively Spliced Exons

The unc-52 gene in Caenorhabditis elegans produces several large proteins that function in the basement membrane underlying muscle cells. Mutations in this gene result in defects in myofilament assembly and in the attachment of the myofilament lattice to the muscle cell membrane. The st549 and ut111 alleles of unc-52 produce a lethal (Pat) terminal phenotype whereas the e444, e669, e998, e1012 and e1421 mutations result in viable, paralyzed animals. We have identified the sequence alterations responsible for these mutant phenotypes. The st549 allele has a premature stop codon in exon 7 that should result in the complete elimination of unc-52 gene function, and the ut111 allele has a Tc1 transposon inserted into the second exon of the gene. The five remaining mutations are clustered in a small interval containing three adjacent, alternatively spliced exons (16, 17 and 18). These mutations affect some, but not all of the unc-52-encoded proteins. Thirteen intragenic revertants of the e669, e998, e1012 and e1421 alleles have also been sequenced. The majority of these carry the original mutation plus a G to A transition in the conserved splice acceptor site of the affected exon. This result suggests that reversion of the mutant phenotype in these strains may be the result of exon-skipping.     

Natural killer gene complex (Nkc) allelic variability in inbred mice: evidence for Nkc haplotypes

Allelic variability for mouse Chromosome 6 Nkc loci was assessed in 22 common laboratory strains of mice using selected natural killer gene complex (Nkc)-linked sequence tagged site markers. Most Nkc markers distinguished three or more alleles for a particular locus in the assessed mouse strains. Nkc locus alleles were highly conserved among genealogically related inbred strains, whereas far less similarity was observed among unrelated strains. Concurrent strain-to-strain comparisons for all Nkc-linked loci revealed common and uncommon Nkc haplotypes, including some that were likely recombinant. Nkc allele and haplotype assignments in inbred mouse strains and correlation with phenotypic traits should facilitate positional gene cloning strategies for unknown Nkc-linked trait modification loci.     

Molecular tagging and candidate gene analysis of the high gamma-tocopherol trait in safflower (<Emphasis Type="Italic">Carthamus tinctorius</Emphasis> L.)

Genetic control of the synthesis of high gamma-tocopherol (gamma-T) content in the seed oil of safflower (Carthamus tinctorius L.) and development of highly reliable molecular markers for this trait were determined through molecular tagging and candidate gene approaches. An F2 population was developed by crossing the high gamma-T natural mutant IASC-1 with the CL-1 line (standard, high alpha-T profile). This population segregated for the partially recessive gene Tph2. Bulked segregant analysis with random amplified polymorphic DNA (RAPD) and microsatellite (SSR) markers revealed linkage of eight RAPD and one SSR marker loci to the Tph2 gene and allowed the construction of a Tph2 linkage map. RAPD fragments closest to the Tph2 gene were transformed into sequence-characterized amplified region markers. A gamma-T methyltransferase (gamma-TMT) locus was found to co-segregate with Tph2. The locus/band was isolated, cloned and sequenced and it was confirmed as a gamma-TMT gene. A longer partial genomic DNA sequence from this gene was obtained. IASC-1 and CL-1 sequence alignment showed one non-synonymous and two synonymous nucleotide mutations. Intron fragment length polymorphism and insertion-deletion markers based on the gamma-TMT sequence diagnostic for the Tph2 mutation were developed and tested across 22 safflower accessions, cultivars, and breeding lines. The results from this study provide strong support for the role of the gamma-TMT gene in determining high gamma-T content in safflower and will assist introgression of thp2 alleles into elite safflower lines to develop varieties with improved tocopherol composition for specific market niches.     

A novel point mutation in an acceptor splice site of intron 32 (IVS32 A–12→G) but no exon 3 mutations in the glycogen debranching enzyme gene in a homozygous patient with glycogen storage disease type IIIb

Genetic deficiency of the glycogen-debranching enzyme (debrancher) causes glycogen storage disease type III (GSD III), which is divided into two subtypes: IIIa and IIIb. In GSD IIIb, glycogen accumulates only in the liver, whereas both liver and muscles are involved in GSD IIIa. The molecular basis for the differences between the two subtypes has not been fully elucidated. Recently, mutations in exon 3 of the debrancher gene were reported to be specifically associated with GSD IIIb. However, we describe a homozygous GSD IIIb patient without mutations in exon 3. Analysis of the patient’s debrancher cDNA revealed an 11-bp insertion in the normal sequence. An A to G transition at position –12 upstream of the 3′ splice site of intron 32 (IVS 32 A^–12→G) was identified in the patient’s debrancher gene. No mutations were found in exon 3. Mutational analysis of the family showed the patient to be homozygous for this novel mutation as well as three polymorphic markers. Furthermore, the mother was heterozygous and the parents were first cousins. The acceptor splice site mutation created a new 3′ splice site and resulted in insertion of an 11-bp intron sequence between exon 32 and exon 33 in the patient’s debrancher mRNA. The predicted mutant enzyme was truncated by 112 amino acids as a result of premature termination. These findings suggested that a novel IVS 32 A^–12→G mutation caused GSD IIIb in this patient. Received: 1 August 1997 / Accepted: 22 September 1997     

Four novel PEPD alleles causing prolidase deficiency.

Mutations at the PEPD locus cause prolidase deficiency (McKusick 170100), a rare autosomal recessive disorder characterized by iminodipeptiduria, skin ulcers, mental retardation, and recurrent infections. Four PEPD mutations from five severely affected individuals were characterized by analysis of reverse-transcribed, PCR-amplified (RT-PCR) cDNA. We used SSCP analysis on four overlapping cDNA fragments covering the entire coding region of the PEPD gene and detected abnormal SSCP bands for the fragment spanning all or part of exons 13-15 in three of the probands. Direct sequencing of the mutant cDNAs showed a G-->A, 1342 substitution (G448R) in two patients and a 3-bp deletion (delta E452 or delta E453) in another. In the other two probands the amplified products were of reduced size. Direct sequencing of these mutant cDNAs revealed a deletion of exon 5 in one patient and of exon 7 in the other. Intronic sequences flanking exons 5 and 7 were identified using inverse PCR followed by direct sequencing. Conventional PCR and direct sequencing then established the intron-exon borders of the mutant genomic DNA revealing two splice acceptor mutations: a G-->C substitution at position -1 of intron 4 and an A-->G substitution at position -2 of intron 6. Our results indicate that the severe form of prolidase deficiency is caused by multiple PEPD alleles. In this report we attempt to begin the process of describing these alleles and cataloging their phenotypic expression.     

Mutations in TRIOBP, which encodes a putative cytoskeletal-organizing protein, are associated with nonsyndromic recessive deafness

In seven families, six different mutant alleles of TRIOBP on chromosome 22q13 cosegregate with autosomal recessive nonsyndromic deafness. These alleles include four nonsense (Q297X, R788X, R1068X, and R1117X) and two frameshift (D1069fsX1082 and R1078fsX1083) mutations, all located in exon 6 of TRIOBP. There are several alternative splice isoforms of this gene, the longest of which, TRIOBP-6, comprises 23 exons. The linkage interval for the deafness segregating in these families includes DFNB28. Genetic heterogeneity at this locus is suggested by three additional families that show significant evidence of linkage of deafness to markers on chromosome 22q13 but that apparently have no mutations in the TRIOBP gene.     

Isolation of polymorphic microsatellite markers for Begonia sutherlandii Hook. f.

Seven polymorphic microsatellite loci have been characterized for investigating population structure in the patchily distributed herb Begonia sutherlandii. Two loci (BSU3 and BSU4) exhibited population specific null alleles; primer redesign and allele sequencing for one of these loci showed two transition mutations in the original primer site. Two loci exhibited imperfect repeat polymorphisms due to single base pair indels in the flanking region (locus BSU6) and in the microsatellite region itself (BSU7). Transversion mutations were also found in the microsatellite region of locus BSU7. The remaining three loci amplified in all individuals tested and appeared to conform to a simple stepwise mutation pattern.     

Genetic Interactions at the Fla10 Locus: Suppressors and Synthetic Phenotypes That Affect the Cell Cycle and Flagellar Function in Chlamydomonas Reinhardtii

Through the isolation of suppressors of temperature-sensitive flagellar assembly mutations at the FLA10 locus of Chlamydomonas reinhardtii, we have identified six other genes involved in flagellar assembly. Mutations at these suppressor loci, termed SUF1-SUF6, display allele specificity with respect to which fla10- mutant alleles they suppress. An additional mutation, apm1-122, which confers resistance to the plant herbicides amiprophos-methyl and oryzalin, was also found to interact with mutations at the FLA10 locus. The apm1-122 mutation in combination with three fla10- mutant alleles results in synthetic cold-sensitive cell division defects, and in combination with an additional pseudo-wild-type fla10- allele yields a synthetic temperature-sensitive flagellar motility phenotype. Based upon the genetic interactions of these loci, we propose that the FLA10 gene product interacts with multiple components of the flagellar apparatus and plays a role both in flagellar assembly and in the cell cycle.     

Discovery and assay of single-nucleotide polymorphisms in barley (Hordeum vulgare)

The least ambiguous genetic markers are those based on completely characterized DNA sequence polymorphisms. Unfortunately, assaying allele states by allele sequencing is slow and cumbersome. The most desirable type of genetic marker would be unambiguous, inexpensive to assay and would be assayable singly or in parallel with hundreds of other markers (multiplexable). In this report we sequenced alleles at 54 barley (Hordeum vulgare ssp. vulgare) loci, 38 of which contained single-nucleotide polymorphisms (SNPs). Many of these 38 loci contained multiple polymorphisms, and a total of 112 polymorphisms were scored in five barley genotypes. The polymorphism data set was analyzed both by using the individual mutations as cladistic characters and by reducing data for each locus to haplotypes. We compared the informativeness of these two approaches by consensus tree construction and bootstrap analysis. Both approaches provided similar results. Since some of the loci sequenced contained insertion/deletion events and multiple point mutations, we thought that these multiple-mutated loci might represent old alleles that predated the divergence of barley from H. spontaneum. We evaluated sequences from a sample of H. spontaneum accessions from the Eastern Mediterranean, and observed similar alleles present in both cultivated barley and H. spontaneum, suggesting either multiple domestication events or multiple transfers of genes between barley and its wild ancestor.     

Identification of microsatellite markers from Cicer reticulatum: molecular variation and phylogenetic analysis

Microsatellite sequences were cloned and sequenced from Cicer reticulatum, the wild annual progenitor of chickpea (C. arietinum L.). Based on the flanking sequences of the microsatellite motifs, 11 sequence-tagged microsatellite site (STMS) markers were developed. These markers were used for phylogenetic analysis of 29 accessions representing all the nine annual Cicer species. The 11 primer pairs amplified distinct fragments in all the annual species demonstrating high levels of sequence conservation at these loci. Efficient marker transferability (97%) of the C. reticulatum STMS markers across other species of the genus was observed as compared to microsatellite markers from the cultivated species. Variability in the size and number of alleles was obtained with an average of 5.8 alleles per locus. Sequence analysis at three homologous microsatellite loci revealed that the microsatellite allele variation was mainly due to differences in the copy number of the tandem repeats. However, other factors such as (1) point mutations, (2) insertion/deletion events in the flanking region, (3) expansion of closely spaced microsatellites and (4) repeat conversion in the amplified microsatellite loci were also responsible for allelic variation. An unweighted pairgroup method with arithmetic averages (UPGMA)-based dendrogram was obtained, which clearly distinguished all the accessions (except two C. judaicum accessions) from one another and revealed intra- as well as inter-species variability in the genus. An annual Cicer phylogeny was depicted which established the higher similarity between C. arietinum and C. reticulatum. The placement of C. pinnatifidum in the second crossability group and its closeness to C. bijugum was supported. Two species, C. yamashitae and C. chorassanicum, were grouped distinctly and seemed to be genetically diverse from members of the first crossability group. Our data support the distinct placement of C. cuneatum as well as a revised classification regarding its placement.     

Isolation and characterization of 149 novel microsatellite DNA markers for striped bass,Morone saxatilis,and cross‐species amplification in white bass,Morone chrysops,and their hybrid

To support detailed genetic analysis of striped bass (Morone saxatilis) and white bass (Morone chrysops), we isolated 153 microsatellite loci from repeat‐enriched striped bass DNA libraries. Of these, 147 markers amplified in striped bass (average 4.7 alleles per locus) and 133 in white bass (average 2.2 alleles per locus). One hundred twenty‐two markers amplified in their hybrid. Development of new microsatellite markers will facilitate evaluations of genetic structure in wild populations and will support pedigree analysis and linkage mapping for selective breeding.     

Conservation of (GA)n microsatellite loci between Quercus species

Microsatellite (simple sequence repeat, SSR) amplification was performed in eight different members of the Fagaceae family by using sets of primers developed from sessile oak, Quercus petraea . In total, 136 cases of heterologous amplification were carried out, and 66% resulted in interpretable amplification products. From these, 12 PCR amplification products were sequenced and all 12 contained a sequence homologous to the original locus from Q. petraea . Although SSR primers worked even across different genera, with increasing evolutionary distance there was a clear tendency for decreasing ability to successfully amplify loci and a decreasing proportion of polymorphism amongst those markers which could be amplified. Two of the loci, ssrQpZAG46 and ssrQpZAG110, were polymorphic in all Quercus species tested. Only at one locus, ssrQpZAG58, a specific PCR product could be amplified in all species analysed. For four loci found in two species, we observed significant interspecies differences in the size range of the amplified alleles. Sequence analysis of two alleles showed that the size differences are not only due to variations in the number of (GA) repeats but also to an insertion of approximately 80 nucleotides in the flanking region. Our findings prove the usefulness of SSR markers within and amongst closely related genera of plants.     

A single nucleotide polymorphism in an exon dictates allele dependent differential splicing of episialin mRNA

The episialin gene (MUC1) encodes an epithelial mucin containing a variable number of repeats with a length of twenty amino acids, resulting in many different alleles that can be subdivided into two size classes. The episialin pre-mRNA uses either one of two neighbouring splice acceptor sites for exon 2, which mainly encodes the repeats. Using the genetic polymorphism of the episialin gene to identify different alleles, we show here that the splice site recognition is allele dependent and is based on a single A/G nucleotide difference in exon 2 eight nucleotides downstream of the second splice acceptor site. Transfection experiments confirm that this polymorphic nucleotide regulates the splice site selection. The identity of this nucleotide is in most cases correlated with one of the size classes of the alleles, indicating that mutations altering the number of repeats seldom arise by unequal cross-over between the repeat regions.     

种间转移扩增筛选仙湖苏铁微卫星位点及其遗传多样性研究

运用刺叶苏铁、葫芦苏铁、海南苏铁的SSR引物,在仙湖苏铁中进行种间转移扩增,筛选得到7对引物能扩增出清晰的特异带,其中3对引物的扩增产物具有多态性.为验证微卫星的真实性,扩增产物切胶回收后克隆测序.结果表明：重复单元的数目变化是扩增片段长度多态性的主要来源.运用筛选出的3对 SSR标记对4个仙湖苏铁野生种群进行遗传结构研究,等位基因数从2~5,杂合度从0．000~0．667,期望杂合度为0．000~0．610.种群两两遗传分化系数从0~0．382.总体上仙湖苏铁遗传多样性水平低,而种群间遗传分化显著.STRUCTURE分析结果表明,4个野生种群可被分配到3个假想的遗传簇.BOTTLENECK 分析结果表明种群近期没有遭遇瓶颈效应.     

MTHFR及PAI基因多态性与新生儿早产易感性的关系

目的:探讨MTHFR基因、PAI-1基因多态性与新生儿早产的关系。方法:选自2014年1月-2015年1月期间我院住院患儿285例并分为四组。抽取研究对象静脉血进行目的基因MTHFR基因C677T、PAI基因的提取、扩增及检测。结果:MTHFR基因C677T扩增片段198 bp,经限制性内切酶作用后形成野生CC型(片段198 bp)、纯合子突变TT型(175 bp、23 bp)以及杂合子突变CT型(23 bp、175 bp以及198 bp);PAI基因扩增片段142 bp,经限制性内切酶作用后形成三中基因型,分别为4G/4G型(96 b p、46 bp)、5G/5G型(22 bp、46 bp以及74 bp)以及4G/5G型(22 bp、46 bp、74 bp以及96 bp)。早产儿童与足月儿童MTHFR基因67 7位点T等位基因分布差异显著(P0.05),早产儿童与足月儿童PAI基因启动子675位点4G等位基因分布无显著性差异(P0.05)。结论:早产发生的易感性与多方面因素有关,其中遗传因素方面MTHFR基因677位点T等位基因多态性可能与新生儿早产相关,而PAI基因启动子675位点基因的多态性与新生儿早产的发生无显著相关。     

Evaluating the utility of microsatellites for investigations of autopolyploid taxa

Autopolyploid taxa present numerous challenges for population genetic analyses due to difficulties determining allele dosage. Dosage ambiguity hinders accurate assessment of allele frequencies, multilocus genotypes (MLGTs), as well as levels and patterns of clonality. The pervasiveness of polyploidy in the evolutionary history of plant taxa makes this a recurring problem. Whereas diploidization of loci may occur over time, duplication of at least some loci is still frequently evident. Fortunately, with high-quality allozyme gels, it is possible to accurately infer allele dosage and, thus, determine exact MLGTs. However, accurately assessing dosage of microsatellite peaks is nearly impossible when studying wild populations with a large number of alleles per locus. Even if precise knowledge of genotypes is not required, for comparable numbers of alleles per locus and loci, the number of "phenotypes" is always lower with microsatellites than allozymes due to the inability to assess allele dosage. Microsatellite loci typically have more alleles per locus relative to allozymes although fewer loci are generally employed. Here, we present a mathematical model for comparing the relative utility of simple sequence repeat (SSR) versus allozyme markers to discriminate MLGTs. For example, the average plant allozyme study (2.6 alleles per locus, 10 polymorphic loci) has better discriminating power than SSR markers with 10 alleles at each of 3 loci, 9 alleles at 4 loci, 6 alleles at 5 loci, 5 alleles at 6 loci, and 4 alleles at 8 loci, demonstrating the value of assessing the relative discriminating power of these markers.     

The conversion of RFLP markers to allele specific amplicons linked to QTLs governing malting quality in barley

Application of marker-assisted selection with RFLP based markers has been constrained by high cost and time requirements in situations involving a large number of plants. RFLP markers mapped on a Harrington/TR306 population have been identified elsewhere as linked to quantitative trait loci (QTL) governing malting quality. The probes ABG610, ABC622, as well as probes for the Nar1, Amy1 and Nar7 were sequenced and locus specific primers developed. These locus specific primers were applied to genomic DNA from both Harrington and TR306. Sequence analysis of the resultant monomorphic fragments revealed sequence divergence for the Xabg610, Xabc622, Amy1 and Nar1 loci, but not for the Nar7 locus. Application of a set of Hor2 primers to genomic DNA from the barley lines Harrington and TR306 led to the direct amplification of codominant alleles. Allele-specific primers were designed based on the sequence divergence identified among the Xabg610, Xabc622 and Nar1 alleles. Amplification conditions were optimized for each of these alleles such that only the favourable allele from Harrington was amplified. The usefulness of these primers for selecting Harrington alleles was demonstrated by their failure to amplify the corresponding alleles from the lines, Sterling, Stella and WM872. The Amy1 allele-specific amplicon was only capable of differentiating this locus between Harrington and TR306. The conversion of these markers into PCR amplifiable, allele-specific amplicons would greatly facilitate their application to barley breeding programs.