Chromosomal deletions in Streptomyces griseus that remove the afsA locus

We have recently constructed a physical map of the Streptomyces griseus 2247 genome using the restriction enzymes AseI and DraI, which revealed that this strain carries a 7.8 Mb linear chromosome. Based on this map, precise macrorestriction fragment and cosmid maps were constructed for both ends of the chromosome, which localized the afsA gene 150 Kb from the left end. Two afsA ⁻ mutants were found to have suffered chromosomal deletions that removed the afsA locus. The sizes of the deletions were 20 and 130 Kb at the right end and 180 and 350 kb at the left end, respectively. Hybridization experiments using cosmids carrying a deletion endpoint indicated that the ends of the chromosome in the mutants were fused to form a circular chromosome. Received: 29 July 1996 / Accepted: 27 August 1996     

拟南芥黄心突变体yh基因的图位克隆及分析

在研究光合作用相关基因的过程中,获得了一个叶片为黄心(yellow heart,yh)的突变株,与野生型拟南芥(Col 0)相比,其新生叶片发黄,突变表型由隐性单基因控制。采用图位克隆及其精细定位技术,将yh突变基因定位在1号染色体的INS1_55_342与INS1_56_34区间,物理距离约为676 kb。通过测序得知yh在At1g64790第44个内含子剪接处有4个碱基的缺失,导致内含子剪切位点的变化。RT PCR分析显示,该基因表达降低,是At1g64790基因的一个新等位突变。研究表明,yh突变体与叶绿体的发育相关,可为进一步探究植物叶绿体和叶片发育机制提供新的遗传材料。     

Expression of the tyrosinase-encoding gene in a colorless melanophore mutant of the medaka fish, Oryzias latipes

In the medaka fish Oryzias latipes many mutants for body colors have been isolated. Among them, a colorless melanophore mutant b, carrying b alleles homozygously, has pigmented black eyes but orange-colored skin with amelanotic melanophores, suggesting the presence of a tissue-specific mechanism of melanin formation. To cast light on the molecular basis of the mechanism, we have cloned cDNAs for tyrosinase (Tyr), a key enzyme in melanin biosynthesis, from the wild-type (wt) fish. DNA sequence analysis revealed that all clones encode a protein of 540 amino acids, having five potential glycosylation sites and two copper-binding sites that are characteristic features of Tyr. Genomic DNA blot analysis disclosed that the Tyr gene is present as a single copy in the fish genome. Using a cDNA clone as a probe, RNA blot analysis was carried out. In the wt, the 2.2-kb Tyr mRNA was expressed in eyes and skin but not in liver, corresponding to tissue-specific melanin formation. In the b mutant, contrary to expectation, the mRNA was detected not only in eyes but also in amelanotic skin. Therefore, pigmentation of the skin controlled by b is not directly related to expression of the Tyr gene.     

Chromosome landing at the barley Rar1 locus

The barley Rar1 gene is an essential component of the race-specific, Mla-12-specified powdery mildew resistance reaction. As part of a map-based cloning strategy designed to isolate Rar1, five barley yeast artificial chromosomes (YACs) have been identified, ranging in size from 300 to 1100 kb. PCR-based YAC end-specific markers have been established and were employed to construct a local YAC contig. Four out of five YAC clones were found to be non-colinear with the source DNA. High-resolution genetic mapping of the YAC ends demonstrated that the set of five overlapping YAC clones encompasses the barley Rar1 gene. Received: 9 June 1998 / Accepted: 15 July 1998     

Sequence comparison of the Caenorhabditis elegans dpy-13 and col-34 genes, and their deduced collagen products

A 2232-nucleotide sequence spanning the col-34 gene from the nematode, Caenorhabditis elegans, is presented. This gene, which encodes a collagen protein (Clg), is transcribed from right to left with respect to the genetic map, and convergently with the nearby dpy-13 gene which also encodes a Clg. Both col-34 and dpy-13 have 5'-flanking elements in common with each other and also with other nematode Clg-encoding genes (clg). One element, variants of which are shared by col-7, col-19 and dpy-13, is predicted to be a target for a number of regulatory molecules, possibly including the ceh-18 product, a nematode POU-domain protein. The deduced amino acid sequence of Col-34 has a high degree of homology with the Dpy-13 collagen, although there are significant differences. In particular, one region of Dpy-13, which is predicted to have secondary structure different from Col-34, is altered by the recessive dpy-13(e225) mutation.     

Approaches to lipid metabolism gene identification and characterization in the postgenomic era

The availability of genomic resources has already had a tremendous impact on biomedical research. In this review, we describe how whole genome sequence and high-throughput functional genomics projects have facilitated the identification and characterization of important genes in lipid metabolism and disease. We review key approaches and lipid genes identified in the first years of this century and discuss how genomic resources are likely to streamline gene identification and functional characterization in the future.     

Comparative transcription map of the wobbler critical region on mouse chromosome 11 and the homologous region on human chromosome 2p13-14

Background  

To support the positional cloning of the mouse mutation wobbler (wr ) the corresponding regions on human Chr2p13-14 and mouse Chr11 were analyzed in detail and compared with respect to gene content, order, and orientation.     

Construction of a Bacterial Artificial Chromosome Library of Phytophthora infestans and Transformation of Clones into P. infestans

A bacterial artificial chromosome (BAC) library of Phytophthora infestans was constructed in a derivative of pBELOBACII that had been modified by adding a npt selectable marker gene for transforming P. infestans. A total library of 8 genome equivalents was generated and 16,128 clones with inserts averaging 75 kb (4.9 genome equivalents) were individually picked and stored as an arrayed library in microtiter plates. This coverage was confirmed by screening the library for 11 DNA loci by colony hybridization and by polymerase chain reaction of DNA pools. Transformation of P. infestans with BAC clones containing inserts of 93 to 135 kb was demonstrated. The efficiency of transformation with most BACs was noticeably higher than that with smaller plasmids. Detailed analyses of transformants obtained with a 102-kb BAC indicated that entire inserts were present in about one-quarter of the transformants.     

High-resolution linkage mapping for the non-brittle rachis locus btr1 in cultivated × wild barley (Hordeum vulgare)

Brittle rachis is an important trait to elucidate the domestication process in barley. Brittle rachis in wild barley (Hordeum vulgare ssp. spontaneum (C. Koch.) Thell) is controlled by two dominant complementary genes, Btr1 and Btr2. Cultivated barley (H. vulgare L. ssp. vulgare) lost the brittle rachis character during domestication as a result of mutation at the Btr1 or Btr2 locus. In this study, a high-resolution map of the btr1 locus was constructed using an F₂ population of cultivar (cv. ‘Kanto Nakate Gold’) × wild barley (line OUH602). We cloned and sequenced 26 AFLP markers linked with the btr1 and btr2 loci. Ten converted STS markers were located on the short arm of chromosome 3H only, and at least 9 of the 10 STS markers were allelic with their original AFLPs. Efficient conversion of co-dominant STS markers using BAC clones was successful. No suppression of recombination was observed in the btr1 region even though wild barley was used as one of the parents. Initial results of BAC screening confirmed the resolution power of the developed high resolution map.     

Mapping of mouse carbonic anhydrase-3, Car-3: another locus in the homologous region of mouse chromosome 3 and human chromosome 8

At least six separate genes determining tissue- and organelle-specific isoforms of carbonic anhydrase are known. We have determined the chromosome location of one of these genes, carbonic anhydrase-3 (Car-3), in the mouse and carried out a linkage analysis of Car-1, Car-2, and Car-3. Car-3 has been assigned to band 3A2 by in situ hybridization. We identified a PstI restriction fragment length polymorphism between Mus spretus and Mus mus domesticus and, by using an interspecific backcross, showed that Car-3 is 2.4 +/- 1.7% SE from both Car-1 and Car-2, calculating genetic distance as percentage recombination. No recombinants were found between Car-1 and Car-2 in 100 backcross offspring, and when these data are combined with earlier results, these two loci are estimated to be 1.2 cM from each other at the 95% confidence interval. The three homologous carbonic anhydrase loci in man had earlier been assigned to 8q22, and the finding of linkage of Car-3 to Car-1 and Car-2 in the mouse adds another locus to the conserved segments on mouse chromosome 3 and human chromosome 8.     

基因克隆的方法进展

基因克隆一般分为定位克隆和表型克隆。表型克隆进展较快,主要有消减杂交、代表性差异分析法、mRNA差异显示、DNA转染法及抑制消减杂交法。抑制消减杂交法是1996年报道的一种表型克隆的新方法,是目前寻找差异表达基因的较有效方法,较过去的方法有许多先进之外。本文对此方法的原理及应用作一详细介绍,并与其他方法作简单比较。     

Genetic and physical localization of the root-knot nematode resistance locus Mi in tomato

As part of a map-based cloning strategy designed to isolate the root-knot nematode resistance gene Mi, tomato F2 populations were analyzed in order to identify recombination points close to this economically important gene. A total of 21 089 F2 progeny plants were screened using morphological markers. An additional 1887 F2 were screened using PCR-based flanking markers. Fine-structure mapping of recombinants with newly developed AFLP markers, and RFLP markers derived from physically mapped cosmid subclones, localized Mi to a genomic region of about 550 kb. The low frequency of recombinants indicated that recombination was generally suppressed in these crosses and that crossovers were restricted to particular regions. To circumvent this problem, a population of Lycopersicon peruvianum, the species from which Mi was originally introgressed, that was segregating for resistance was developed. Screening of this population with PCR, RFLP and AFLP markers identified several plants with crossovers near Mi. Recombination frequency was approximately eight-fold higher in the Mi region of the L. peruvianum cross. However, even within the wild species cross, recombination sites were not uniformly distributed in the region. By combining data from the L. esculentum and L. peruvianum recombinant analyses, it was possible to localize Mi to a region of the genome spanning less than 65 kb. Received: 15 July 1997 / Accepted: 1 October 1997     

Mapping in the mouse of the region homologous to the rat growth and reproduction complex (grc)

Offprint requests: J. Klein.     

The effect of mutant genes at the r, rb, rug3, rug4, rug5 and lam loci on the granular structure and physico-chemical properties of pea seed starch

The granular structure and gelatinisation properties of starches from a range of pea seed mutants were studied. Genes which affect the supply of substrate during starch synthesis (rb, rug3, rug4) affected the total crystallinity and possibly increased the content of A polymorphs in the starch. Conversely, genes directly affecting the synthesis of starch polymers (r, rug5, lam) increased the content of B polymorphs, but had a minimal effect on total crystallinity. During gelatinisation, starches from the rb, rug3, rug4 and lam mutants had narrow endothermic peaks which were similar to starch from the wild-type, although all the starches had different peak temperatures and enthalpy changes. Starches from r and rug5 mutants were very different to all other starches, having a very wide transition during gelatinisation. In addition, the amylopectin in starch from these mutants had altered chain lengths for those parts of the polymer which form the ordered structures in the granule.     

The vestigial locus of Drosophila melanogaster is involved in resistance to inhibitors of dTMP synthesis

The vestigal (vg) gene encodes a nuclear protein which plays a major role in the formation of the wing of Drosophila. Resistance or sensitivity to aminopterin, an inhibitor of the dihydrofolate reductase enzyme in D. melanogaster, seems to be associated with a specific alteration in vg gene function. Wild-type and vg mutant strains selected for growth on increasing concentrations of aminopterin display changes in physiological and biochemical parameters such as viability on normal and aminopterin-containing media, duration of development, wing phenotype, dihydrofolate reductase activity, and cross-resistance to fluorodeoxyuridine (FUdR) and to methotrexate. Our results indicate that the mechanisms of resistance differ in the wild-type and mutant strains. The vg ^83b27 mutant, in which the major part of intron 2 of the vg gene is deleted, is associated with a high rate of resistance to FUdR, an inhibitor of thymidylate synthetase. Moreover, vg ^83b27/vg ^BGheterozygotes, which are wild type when grown on normal medium, display a strong vg phenotype when grown on aminopterin. Our results indicate a role for the vestigial locus in mediating resistance to inhibitors of dTMP synthesis.     

Preferential transposition of aDrosophila P element to the corresponding region of the homologous chromosome

Genetic and physical analyses have demonstrated an intimate interaction or pairing of homologous chromosomes in the nuclei of manyDrosophila cell types. Experiments were performed to determine whether P elements transposing from a given chromosome to its homolog would preferentially insert in the region corresponding to the donor site, perhaps due to such a proximity. AP[lacZ;ry ⁺] element at thecactus locus (35F) on the second chromosome was mobilized and 96 insertions on the homolog were recovered. The distribution of these new insertions was determined by recombination mapping and molecular analysis, and compared with a control set of 93 second-chromosome insertions originating from theX chromosome. A nearly threefold preference was observed for re-insertion in a region of two to three number divisions aroundcactus on the homolog. However, none of these local insertions was actually within 50 kb of the site atcactus corresponding to the starting site. This is in marked contrast to the previously described phenomenon of intrachromosomal local transposition, where the majority of local transpositions are within 10 kb. The data suggest that the mechanisms for intrachromosomal and interchromosomal local transposition are distinct, and are consistent with a model for interchromosomal local transposition involving proximity of homologous chromosomal regions in the nuclei of the germline cells.     

Cartographie de restriction comparée de la séquence d''ADN hautement répétée coupée par Bam HI chez Papio papio et P. cynocephalus

Highly repeated DNA sequences from two baboon species (Papio papio and P. cynocephalus) have been compared using restriction endonucleases. The two species share a 343 base pairs tandemly repeated DNA, that is cut once by Bam HI. Papio cynocephalus differs from P. papio by loss of an EcoRI star site in the repeated sequence.     

Identification of 10 882 porcine microsatellite sequences and virtual mapping of 4528 of these sequences

A total of 10 882 porcine microsatellite repeats were identified in genomic shotgun sequences from the Sino-Danish Pig Genome Sequencing Consortium (http://www.piggenome.dk). Of these, 4528 microsatellites were placed on a pig-human comparative map by blast analysis of porcine sequences against the human genome (blast cut-off threshold =1 x 10(-5)). All microsatellite sequences placed on the comparative map are accessible at http://www.animalgenome.org/QTLdb/pig.html. These sequences increase the number of identified microsatellites in the porcine genome by several orders of magnitude. They are a new resource of microsatellite sequences for generating markers to be used in linkage studies and in fine mapping and positional cloning of quantitative trait loci.