beta, a novel repetitive DNA element associated with tRNA genes in the pathogenic yeast Candida albicans

We have identified a novel 399 bp repetitive DNA element (which we designate beta  ) 9 bp upstream of a seryl-tRNA_CAG gene in the genome of Candida albicans . There are two copies of the seryl-tRNA_CAG gene, one on each homologue of chromosome VI, and the beta element is found upstream of one copy of the gene in C. albicans strain 2005E. The beta element is not present upstream of either copy of the seryl-tRNA_CAG gene in eight other laboratory strains of C. albicans tested, but was detected in this location in several fresh clinical isolates. Southern blot analysis indicated that there are approximately eight copies of the beta element per diploid C. albicans genome and that it is a mobile element, being present on at least two different chromosomes. Three unique genomic DNA clones containing the beta element were isolated from strain 2005E; in each case, a different tRNA gene was found immediately adjacent to the beta element. Three new tRNA genes from C. albicans have thus been identified: tRNA^Asp, tRNA^Ala and tRNA^Ile. The beta element shows no significant sequence homology to other known prokaryotic or eukaryotic repetitive elements, although an 8 bp repeat at the 3' end of the element is identical to that of the Ty3 retrotransposable element of Saccharomyces cerevisiae . We propose that the beta element is a solo long terminal repeat (LTR) sequence of a Ty3/gypsy-like transposable element in C. albicans that is closely associated with tRNA genes.     

逆转座子样元件Tca1用于白色念珠菌的分类鉴定

已分离到一中等重复序列以及具有逆转座子样结构的元件Tcal(Transposon Candida albicans)。Tcal两端存在两个完全相同同向排列的序列LTR(Long Terminal Repeat)388bp,中间被一5.5kbDNA片段隔开。以alpha及Tcal为探针对40多种来自美国和中国的临床分离到的致病菌株进行杂交分析,根据杂交图谱,这些白色念株菌可被分成若干组,令人感兴趣的是来自同一地区菌种的遗传相关性比来自不同地区的大。比较URA3等基因的杂交结果,支持这种分析。尚未观察到alpha重复序列与其他酵母菌染色体DNA杂交的杂交条带,认为Tcal元件也许与C. albicans的遗传进化及其致病性有某种内在联系。     

Telomeric and dispersed repeat sequences in Candida yeasts and their use in strain identification.

Several different repetitive DNA sequences have been isolated from the pathogenic yeast Candida albicans. These include two families of large dispersed repeat sequences (Ca3, Ca24) and a short (23-bp) tandemly repeated element (Ca7) associated with C. albicans telomeres. In addition, a large subtelomeric repeat (WOL17) has been cloned. DNA fragments containing the telomeric repeats are highly variable among different C. albicans strains. We have shown that the Ca3 repeat is relatively more stable and is suitable for use as a species-specific and strain-specific probe for C. albicans.     

Induced chromosome rearrangements and morphologic variation in Candida albicans.

We have isolated a mutant of Candida albicans that switches between colony morphologies at high frequencies in a strain with several genetic markers. This strain, 1183, has an altered karyotype with two extra chromosomes. The 1183 karyotype is unstable upon passage. Using DNA transformation with the URA3 gene flanked by sequences from the C. albicans repeat sequence 27A, we have marked individual chromosomes of 1183 and 1161, a related smooth, stable strain. Many transformants contained one or more extra chromosomes, ranging in size from 150 kb to 2.1 Mb. Most were less than 800 kb and appeared to be fragments of a single chromosome. All fragments tested derive from one of the two smallest chromosomes. Six of 13 fragments contained the URA3 gene. In some cases, URA3 was located at the end of a fragment with adjacent telomere repeats. The integrated copy of URA3 was unstable in some 1183 transformants. Our results suggest that 1183 has a mutation affecting genomic stability. A connection between karyotypic changes and morphologic variation has been suggested from studies of several C. albicans strains; however, we find that gross karyotypic and morphological changes are separable processes.     

Organization of a Solatium brevidens repetitive sequence related to the TGRI subtelomeric repeats of Lycopersicon esculentum

A species-specific repetitive DNA fragment has been isolated from a genomic library of Solanum brevidens. Sequence analysis revealed a regular organization of three non-homologous subrepeats forming tandemly-arranged composite repetitive units. Interpretation of Southern hybridization patterns based on the known sequence data suggests that the isolated sequence element represents an abundant organization type, although the presence of simple tandem arrays of the subrepeats is also indicated. Seventy-four percent sequence similarity was found between one of the S. brevidens subrepeats (Sb4AX) and a satellite DNA (TGRI) localized as a subtelomeric repeat on almost all Lycopersicon esculentum chromosomes. Insitu hybridization indicated that, similarly to TGRI, the S. brevidens-specific repeats are located at the ends of the arms of several chromosomes. On the basis of the data obtained, a common ancestral sequence can be proposed for the tomato (TGRI) and the S. brevidens (Sb4AX) repeat however, the molecular organization of this element in these two species evolved in a basically different manner.     

利用微分离黑麦1R染色体的体外扩增产物进行染色体着染研究

首先对显微分离出的黑麦（ＳｅｃａｌｅｃｅｒｅａｌｅＬ．）１Ｒ染色体进行了两轮Ｓａｕ３Ａ连接接头介导的ＰＣＲ扩增（ＬＡ＿ＰＣＲ）。经Ｓｏｕｔｈｅｒｎ杂交证实这些染色体扩增片段来源于基因组ＤＮＡ之后,再利用１Ｒ染色体的第二轮扩增产物、黑麦基因组ＤＮＡ、ｒＤＮＡ基因为探针,与其根尖细胞中期分裂相进行染色体原位杂交,发现微分离的１Ｒ染色体体外扩增产物中包含大量的非该染色体特异性重复序列,而其信息量却较黑麦总基因组少;当以适量的黑麦基因组ＤＮＡ进行封阻时,微分离染色体的体外扩增产物成功地被重新定位在中期分裂相的一对１Ｒ染色体上,说明微分离１Ｒ染色体的ＰＣＲ扩增产物中的确包含了该染色体特异性的片段。此外,以从１Ｒ染色体微克隆文库中筛选出的一单、低拷贝序列和一高度重复序列分别为探针,染色体原位杂交检测发现,这一高度重复序列可能为端粒相关序列;而单、低拷贝序列却未检测到杂交信号。这些结果从不同侧面反映出染色体着染技术是证实微分离、微切割染色体的真实来源及筛选染色体特异性探针的有利工具。建立了可供参考的植物染色体着染实验体系,为染色体微克隆技术在植物中的进一步应用提供了便利。     

用于目标序列染色体定位的镜鲤BAC-FISH体系构建

为了构建用于镜鲤(Cyprinus carpio var. specularis)特定基因组序列染色体定位的实验体系, 在细菌人工染色体(Bacterial Artificial Chromosome, BAC)文库筛选池中对已知短序列基因组片段进行PCR扩增, 筛选出包含目标序列的BAC克隆, 提取BAC质粒进行缺刻平移标记制备探针, 开展荧光原位杂交(Fluorescence in situ hybridization, FISH)实验。通过对染色体片前处理、BAC质粒探针制备、C0t-1 DNA封闭基因组重复序列、预杂交、荧光染料选择、信号放大等一系列实验条件和方法的探索优化, 成功实现了目标序列在镜鲤有丝分裂中期染色体上的定位。定位对象既包括在染色体上有单一位点的序列, 如斑马鱼微卫星标记Z6884和Z4268, 也包括在染色体上有多个位点的重复序列, 如黄河鲤性别相关标记CCmf1。来自斑马鱼同一条染色体上的两个微卫星标记被分别定位于镜鲤不同染色体上, 为鲤鱼染色体数目加倍的进化假设提供了一项直接实验证据, 同时将现有遗传连锁图谱与染色体对应起来, 可作为染色体识别和细胞遗传学图谱构建的依据。黄河鲤性别相关重复序列被定位于不少于四条染色体上, 为性别决定相关基因的筛查提供了研究线索。这一BAC-FISH实验体系将成为鲤细胞遗传学图谱构建、基因组进化和比较基因组学研究中的重要研究工具。       

Comparison of the separation of Candida albicans chromosome-sized DNA by pulsed-field gel electrophoresis techniques.

Pulsed-field gel electrophoresis techniques were used to study chromosome-sized DNA molecules of C. albicans. Chromosome-sized DNA of two strains of Candida albicans has been resolved into 8 bands by orthogonal-field-alternation gel electrophoresis (OFAGE). Six bands were observed in chromosomal preparations of C. albicans using field-inversion gel electrophoresis (FIGE). Differences in the electrophoretic mobilities of bands of the strains of C. albicans examined suggests that chromosome-length polymorphisms exist and make it difficult to correlate the banding patterns among strains. These correlations were facilitated, however, by assignment of C. albicans chromosomes by hybridization using a collection of cloned DNA probes specific for each of the 8 observed bands. Southern blotting showed that the 6 FIGE bands consisted of 4 singlets and 2 comigrating doublets, accounting for the 8 bands observed by OFAGE analysis. The agreement between OFAGE and FIGE analysis suggests that the C. albicans haploid genome contains a minimum of 8 chromosomes.     

Segments of chromosomal DNA from Rhynchosciara americana that undergo additional rounds of DNA replication in the salivary gland DNA puffs have only weak ARS activity in yeast

We have constructed a library of recombinant phage containing DNA from salivary gland chromosomes of Rhynchosciara americana. We have isolated phage from this library that carry sequences homologous to cDNA clones that hybridize in situ to the DNA puffs at the polytene chromosome regions C3 and C8. This has enabled us to demonstrate a 16-fold amplification of the genomic DNA sequences at these regions during DNA-puffing. At the C8 site there is a sequence element that has characteristics of 'scrambled' moderately repetitive DNA. This is located within 3 kb from the gene encoding a 1.95-kb mRNA. We have assayed restriction fragments from the two DNA puffs for Ars activity in yeast. The only strong Ars activity is associated with a part of the moderately repetitive DNA element from the C8 puff which is not present at this site in all animals.     

Mer22-related sequence elements form pericentric repetitive DNA families in primates

We describe a novel repetitive DNA element isolated from three primate species belonging to the family Cercopithecidae. The unusually long 2.6-kb repeat unit of this DNA element is present in high copy number in the pericentromeric region of one pair of chromosomes in both baboon and macaque, forming chromosome-specific satellite-like DNA families. Besides these two very closely related species, the novel DNA element was also detected in the more distantly related African green monkey. However, the copy number of the repeat unit in this species is significantly lower than in macaque and baboon. Sequence analysis revealed that the repeat units of the new repetitive element show similarity to the human MER22 repeat and the Y chromosome-specific TTY2 element, which also exhibits retroelement-like features. Database searches indicate that tandemly arranged MER22-related DNA sequences can also be found in human, raising the possibility that these DNA elements may correspond to a novel primate-specific repetitive DNA group. Recent studies indicate that chromosome-specific pericentric repetitive elements, besides their potential involvement in centromere function, also facilitate homolog recognition during meiosis. In addition, rapid expansion of retroelements in the pericentric regions of chromosomes during interspecific hybridization has been described. In light of these data, we hypothesize that the novel repetitive element described here might have been involved in the speciation of the family Cercopithecidae.     

Transposition of a long member of the L1 major interspersed DNA family into the mouse beta globin gene locus.

A long member of the highly repeated long interspersed DNA family L1Md (for L1 in Mus domesticus) has integrated by transposition into a target site which lies between the two adult beta globin genes of mouse. DNA hybridization and nucleotide sequence analysis show that this target site, which is part of the single copy DNA flanking the globin genes, is interrupted by the L1 element in one chromosome but is uninterrupted in both allelic and ancestral chromosomes. Other large DNA rearrangements of the region between the two adult beta globin genes are also associated with these allelic chromosomes, and include insertions or deletions of both single copy DNA and simple and complex repetitive DNA. This has caused extensive reorganization of this intergenic region. However, the distance between the two genes flanking this region remains conserved, suggesting that the spacing of the globin genes may be subject to conservative selection.     

Identification of Bilby, a diverged centromeric Ty1-copia retrotransposon family from cereal rye (Secale cereale L.).

A diminutive rye chromosome (midget) in wheat was used as a model system to isolate a highly reiterated centromeric sequence from a rye chromosome. Fluorescence in situ hybridization (FISH) shows this sequence localized within all rye centromeres and no signal was detected on wheat chromosomes. DNA sequencing of the repetitive element has revealed the presence of some catalytic domains and signature motifs typical of retrotransposon genes and has been called the Bilby family, representing a diverged family of retrotransposon-like elements. Extensive DNA database searching revealed some sequence similarity to centromeric retrotransposons from wheat, barley, and centromeric repetitive sequences from rice. Very low levels of signal were observed when Bilby was used as a probe against barley, and no signal was detected with rice DNA during Southern hybridization. The abundance of Bilby in rye indicates that this family may have diverged from other distantly related centromeric retrotransposons or incorporated in the centromere but rapidly evolved in rye during speciation. The isolation of a rye retrotransposon also allowed the analysis of centromeric breakpoints in wheat-rye translocation lines. A quantitative analysis shows that the breakpoint in IDS.1RL and 1DL.1RS and recombinant lines containing proximal rye chromatin have a portion of the rye centromere that may contribute to the normal function of the centromeric region.     

Chromosomal localization of two novel repetitive sequences isolated from the Chenopodium quinoa Willd. genome

The chromosomal organization of two novel repetitive DNA sequences isolated from the Chenopodium quinoa Willd. genome was analyzed across the genomes of selected Chenopodium species. Fluorescence in situ hybridization (FISH) analysis with the repetitive DNA clone 18-24J in the closely related allotetraploids C. quinoa and Chenopodium berlandieri Moq. (2n = 4x = 36) evidenced hybridization signals that were mainly present on 18 chromosomes; however, in the allohexaploid Chenopodium album L. (2n = 6x = 54), cross-hybridization was observed on all of the chromosomes. In situ hybridization with rRNA gene probes indicated that during the evolution of polyploidy, the chenopods lost some of their rDNA loci. Reprobing with rDNA indicated that in the subgenome labeled with 18-24J, one 35S rRNA locus and at least half of the 5S rDNA loci were present. A second analyzed sequence, 12-13P, localized exclusively in pericentromeric regions of each chromosome of C. quinoa and related species. The intensity of the FISH signals differed considerably among chromosomes. The pattern observed on C. quinoa chromosomes after FISH with 12-13P was very similar to GISH results, suggesting that the 12-13P sequence constitutes a major part of the repetitive DNA of C. quinoa.     

Four types of IS1 with differences in nucleotide sequence reside in the Escherichia coli K-12 chromosome.

The Escherichia coli K-12 chromosome contains six copies of insertion element IS1 at loci is1A-is1F. We determined their nucleotide (nt) sequences and found that they were classified into four types. Two copies of IS1 which flank a chromosomal segment containing the argF gene (IS1B and IS1C) have identical nt sequences. Another identical pair are IS1A and IS1E. Comparison of their nt sequences with the IS1 in plasmid R100 revealed seven nt mismatches for IS1A (or IS1E), two for IS1B (or IS1C), four for IS1D, and 75 for IS1F. The fact that the IS1s flanking the argF segment are identical supports the idea that the segment, together with the particular pair of IS1s, has constituted a composite transposon and transposed after genetic transfer from another bacterial species into E. coli K-12. Duplicated sequences were not observed in the regions flanking each of four copies of IS1, indicating that rearrangements have occurred in these chromosomal regions after IS1 elements had been inserted into several target sites. The four types of IS1 present in the E. coli K-12 chromosome were essentially similar to IS1s in plasmid R100 and in the chromosomes of Shigella strains. This and the above results suggest that they have been transferred horizontally from other Enterobacteriaceae, including Shigella, into E. coli K-12.     

The CACTA transposon Bot1 played a major role in Brassica genome divergence and gene proliferation

We isolated and characterized a Brassica C genome-specific CACTA element, which was designated Bot1 (Brassica oleracea transposon 1). After analysing phylogenetic relationships, copy numbers and sequence similarity of Bot1 and Bot1 analogues in B. oleracea (C genome) versus Brassica rapa (A genome), we concluded that Bot1 has encountered several rounds of amplification in the oleracea genome only, and has played a major role in the recent rapa and oleracea genome divergence. We performed in silico analyses of the genomic organization and internal structure of Bot1, and established which segment of Bot1 is C-genome specific. Our work reports a fully characterized Brassica repetitive sequence that can distinguish the Brassica A and C chromosomes in the allotetraploid Brassica napus, by fluorescent in situ hybridization. We demonstrated that Bot1 carries a host S locus-associated SLL3 gene copy. We speculate that Bot1 was involved in the proliferation of SLL3 around the Brassica genome. The present study reinforces the assumption that transposons are a major driver of genome and gene evolution in higher plants.     

Genomic organization in the flesh fly Sarcophaga bullata

The genome of the flesh fly Sarcophaga bullata has been characterized both cytologically and biochemically. S. bullata has a haploid DNA level of 0.61 picograms which is five times larger than the haploid genome size of Drosophila melanogaster. Reassociation kinetics of Sarcophaga DNA shows that its sequence organization is very similar to that of D. melanogaster in having a very large proportion of single copy DNA (81%) and only small amounts of highly and moderately repetitive DNA (9% and 6%, respectively). cRNAs from all three sequence classes were prepared and their cytological distributions on diploid and polytene cells determined by in situ hybridization. The cytological distribution of the highly repetitive probe was found to be restricted to the centromeric heterochromatin of two of the five autosomes and this sequence class was also found to be markedly underreplicated in polytene foot-pad cells. No highly repetitive DNA was localized on either of the sex chromosomes, but only on the two large centromeric regions of chromosomes C and E. Moderately repetitive DNA was found uniformly distributed on all of the autosomes in both testis and polytene foot-pad squashes. As in the case of the highly repetitive sequence probe, no moderately repetitive DNA was detected on either the X or Y chromosomes. Moderately repetitive DNA in Sarcophaga was also shown to have the Drosophila type pattern of sequence interspersion with a moderately repetitive element of 5,000 nucleotides adjacent to a unique element of greater than 10,000 nucleotides. The Sarcophaga genome is the largest for which this type of interspersion has so far been demonstrated.     

Ty1 and delta elements occur adjacent to several tRNA genes in yeast

A comparative analysis of a number of yeast DNA-pBR322 recombinant plasmids carrying repetitive sequence elements has revealed that Ty1 or delta elements occur in the vicinity of several tRNA genes. Four examples have been characterized in detail: three glutamate tRNA genes and a serine tRNA gene. The tRNAGlu3 genes occupy different chromosomal locations; two of these genes are found adjacent to Ty1 elements, and the third is found adjacent to an independent delta element. A delta unit is also found adjacent to a tRNASer2 gene. Next to one of the tRNAGlu3 genes, the delta element is joined to a truncated sigma element. Junctions between different delta units were characterized by the sequence analysis of two DNA segments that carry no tRNA genes.