Sequence homogenization and chromosomal localization of VicTR-B satellites differ between closely related Vicia species

Satellite sequences of the VicTR-B family are specific for the genus Vicia (Leguminosae), but their abundance varies among the species, being the highest in Vicia sativa and Vicia grandiflora. In this study, we have sequenced multiple randomly cloned VicTR-B fragments from these two species and analyzed their sequence variability, periodicity, and chromosomal localization. We have found that V. sativa VicTR-B sequences are homogeneous with respect to their nucleotide sequences and periodicity (monomers of 38 bp), whereas V. grandiflora repeats are considerably more variable, occurring in at least four distinct sequence subfamilies. Although the periodicity of 38 bp was conserved in most of the V. grandiflora sequences, one of the subfamilies was composed of higher-order repeats of 186 bp, which originated from a pentamer of the basic repeated unit. Individual VicTR-B subfamilies were preferentially located in either intercalary or subtelomeric regions of chromosomes. Interestingly, two V. grandiflora subfamilies with the highest similarity to V. sativa VicTR-B sequences were located in intercalary heterochromatic bands, showing similar chromosomal distribution as the majority of VicTR-B repeats in V. sativa. The other two V. grandiflora subfamilies showing a considerable divergence from V. sativa sequences were found to be accumulated at subtelomeric regions of V. grandiflora chromosomes.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.Communicated by I. Schubert     

Conservation genetics of whales and dolphins

Whales and dolphins (cetaceans) are found in all the world's oceans and in some of the major rivers, yet little is known about the distribution and behaviour of many species. At the same time, cetaceans are under threat from a variety of pressures including direct and indirect takes, pollution, and competition for habitat and prey. To ensure their long-term survival it will be necessary to preserve genetic diversity through the identification and protection of differentiated populations, the assessment of variation within local populations, and through a better understanding of reproductive and dispersal behaviour. The application of molecular genetic techniques is helping to provide answers to some of these previously intractable questions. Early results suggest few consistent patterns. Obvious geographic boundaries correlate to genetic distance in some species, and not in others. Furthermore, morphological variation within species can be fairly extensive without correlating to genetic distance, or relatively minor between morphotypes that are as genetically distinct as some species. These examples emphasize the need for further study.     

DNA methylation specifies chromosomal localization of MeCP2.

MeCP2 is a chromosomal protein that is concentrated in the centromeric heterochromatin of mouse cells. In vitro, the protein binds preferentially to DNA containing a single symmetrically methylated CpG. To find out whether the heterochromatic localization of MeCP2 depended on DNA methylation, we transiently expressed MeCP2-LacZ fusion proteins in cultured cells. Intact protein was targeted to heterochromatin in wild-type cells but was inefficiently localized in mutant cells with low levels of genomic DNA methylation. Deletions within MeCP2 showed that localization to heterochromatin required the 85-amino-acid methyl-CpG binding domain but not the remainder of the protein. Thus MeCP2 is a methyl-CpG-binding protein in vivo and is likely to be a major mediator of downstream consequences of DNA methylation.     

Structure and chromosomal localization of DNA sequences related to ribosomal subrepeats in Vicia faba

Subrepeating sequences of 325 bp found in the ribosomal intergenic spacer (IGS) of Vicia faba and responsible for variations in the length of the polycistronic units for rRNA were isolated and used as probes for in situ hybridization. Hybridization occurs at many regions of the metaphase chromosomes besides those bearing rRNA genes, namely chromosome ends and all the heterochromatic regions revealed by enhanced fluorescence after quinacrine staining. The DNA homologous to the 325 bp repeats that does not reside in the IGS was isolated, cloned and sequenced. It is composed of tandemly arranged 336 bp elements, each comprising two highly related 168 bp sequences. This structure is very similar to that of the IGS repeats and ca. 75% nucleotide sequence identity can be observed between these and the 168 bp doublets. The most obvious difference lies in the deletion, in the former, of a 14 bp segment from one of the two related sequences. It is hypothesized that the IGS repeats are derived from the 336 bp elements and have been transposed to ribosomal cistrons from other genome fractions. The possible relations between these sequences and others with similar structural features found in other species are discussed.by D. Schweizer     

Molecular cloning and chromosomal localization of DNA sequences associated with a human DNA repair gene.

The genes and gene products involved in the mammalian DNA repair processes have yet to be identified. Toward this end we made use of a number of DNA repair-proficient transformants that were generated after transfection of DNA from repair-proficient human cells into a mutant hamster line that is defective in the initial incision step of the excision repair process. In this report, biochemical evidence is presented that demonstrates that these transformants are repair proficient. In addition, we describe the molecular identification and cloning of unique DNA sequences closely associated with the transfected human DNA repair gene and demonstrate the presence of homologous DNA sequences in human cells and in the repair-proficient DNA transformants. The chromosomal location of these sequences was determined by using a panel of rodent-human somatic cell hybrids. Both unique DNA sequences were found to be on human chromosome 19.     

Nucleotide sequence of a highly repeated DNA sequence and its chromosomal localization in Allium fistulosum

A highly repeated DNA sequence with a repeating unit of approximately 380bp was found in EcoRV digests of the total genomic DNA of Allium fistulosum. Three independent clones containing this unit were isolated, and their repeating units sequenced. These units showed more than 94% sequence homology, and the copy number was estimated to be about 2.8×10⁶ per haploid genome. In situ hybridization, with the repeating unit as a probe, and C-banding analyses indicated that the repeated DNA sequence of A. fistulosum is closely associated with the major C-heterochromatin in the terminal regions of all 16 chromosomes at mitotic metaphase. The characters of the repeating unit are similar to those of the A. cepa unit, which is taxonomically closely related to A. fistulosum.     

Phosphorylation of CAP-G is required for its chromosomal DNA localization during mitosis

Condensin is a 5 subunit complex that plays an important role in the structure of chromosomes during mitosis. It is known that phosphorylation of condensin subunits by cdc2/cyclin B at the beginning of mitosis is important for condensin activity, but the sites of these phosphorylation events have not been identified nor has their role in regulating condensin function. Here we identify two threonine residues in the CAP-G subunit of condensin, threonines 308 and 332, that are targets of cdc2/cyclin B phosphorylation. Mutation of these threonines to alanines results in defects in CAP-G localization with chromosomes during mitosis. These results are the first to identify phosphorylation sites within the condensin complex that regulate condensin localization with chromosomal DNA.     

Schistosoma mansoni: chromosomal localization of DNA repeat elements by in situ hybridization using biotinylated DNA probes

Localization of the SM alpha family of repeated DNA and the rDNA repeat on the chromosomes of Schistosoma mansoni by in situ hybridization is presented. Biotinylated DNA was hybridized to target chromosomes and hybridization was detected using either alkaline phosphatase-labeled avidin or fluorescein-labeled avidin and biotinylated anti-avidin antibody. Hybridization detection using a fluorescein conjugate was more specific and sensitive with less background noise than detection with alkaline phosphatase conjugates. SM alpha hybridizing sequences were found dispersed throughout the genome, hybridizing to the sex chromosomes and autosomes. The SM alpha probe showed specific hybridization to the euchromatic gap region within the large heterochromatic block of the short arm of the W chromosome. This specific hybridization coupled with the lack of chiasma formation in this region of the ZW bivalent (presumably due to the heterochromatinization of this region) may explain the pattern of sex-specific hybridization reported for the SM alpha family. The rDNA repeat was localized to the secondary constriction of the short arm of chromosome 3. Specifically, the rDNA probe hybridized with the stalk of the secondary constriction and with parts of both side regions, the satellite and the short arm proper.     

Sequence organization and evolution,in all extant whalebone whales,of a DNA satellite with terminal chromosome localization

A heavy (GC rich) DNA satellite with terminal chromosomal localization is characteristic for all mysticete (whalebone whale) genomes. Sequences of 58 repeats of the satellite were compared in all ten extant mysticete species. In three families comprising eight species, the typical repeat length was 422(421) bp. In two species, the northern right whale and the bowhead, of family Balaenidae (right whales) the repeats were much longer, typically ca. 900 and ca. 1200 bp. In all species the repeats were composed of a unique portion of constant length (212/211 bp), and a subrepeat portion, the length of which was variable. The evolutionary rigidity of the unique portion of the repeat is contrasted by the pronounced length variability of the subrepeat portion. The subrepeat portion consists essentially of 6 bp motifs, such that length differences are usually in multiples of 6 bp. The motif TTAGGG constituted 35%–50% of the subrepeats. Comparison between the unique portion of the 58 sequenced repeats revealed that the repeats divided into two primary groups, one comprising the two balaenids, the other including the eight remaining species. The mean difference between the two groups averaged 8.4%. In this sequence comparison the repeats of the pygmy right whale constituted a group that was separated from repeats of the other species. In all other cases repeats were intermingled to some extent between species. Comparison of individual repeats suggests that the unique portion evolves in concert, at a slow rate. A neighborjoining comparison between the consensuses of all species suggests that the unique portion of the repeats evolves at a somewhat different rate in different lineages.     

Preliminary karyotype and chromosomal localization of ribosomal DNA sites in white spruce using fluorescence in situ hybridization.

We have localized the major ribosomal DNA (rDNA) loci on metaphase chromosomes and in interphase nuclei of white spruce (2n = 24) by fluorescence in situ hybridization. Hybridization sites of the biotin-labelled rDNA probe were detected using antibody-fluorochrome conjugates and a confocal laser scanning microscope. White spruce has at least 12, and possibly as many as 14, rDNA sites, 1 site present on each of seven separate chromosome pairs. This is one of the highest numbers of rDNA loci yet reported among plant species. The position of the rDNA loci together with secondary constriction patterns permit, for the first time, all homologous pairs of white spruce chromosomes to be distinguished. We discuss the application of molecular cytogenetics in studies relating to the organization and evolution of DNA sequences within conifer genomes.     

The chromosomal localization of a heavy satellite DNA in the testis of Plethodon c. cinereus

When DNA from blood or liver of Plethodon c. cinereus is centrifuged to equilibrium in cesium chloride it separates out into 2 components. The smaller or satellite component is relatively rich in G + C and is therefore heavy, and it amounts to about 2% of the total DNA. The heavy satellite does not include the ribosomal cistrons, and it is unrelated to the nucleolar organizer. When squash preparations of cells from the testis of P. c. cinereus are incubated in synthetic E³RNA complementary to the satellite DNA, the RNA anneals specifically to the centromeric heterochromatin of spermatogonia, spermatocytes, and spermatids, and to the centromeric regions of all discernible chromosomes. RNA/DNA hybrids were located by autoradiography. H³RNA complementary to the major component of the DNA anneals to all nuclei and to all parts of the chromosomes. H³RNA complementary to nucleolar DNA from Xenopus laevis anneals specifically to the chromatin associated with nucleoli in nuclei at various stages of the meiotic divisions. The nature of the centromeric heterochromatin and its role in the meiotic divisions are discussed.     

Isolation and chromosomal localization of genomic DNA sequences coding for cytoplasmic superoxide dismutase from Drosophila melanogaster

A synthetic oligodeoxynucleotide 18-mer probe derived from the amino acid sequence of Drosophila melanogaster cytoplasmic superoxide dismutase (cSOD) was used to screen a D. melanogaster genomic library. One of the positive clones maps by in situ hybridization to position 68A8-9 on the left arm of polytene chromosome 3, the region to which cSOD mutants have previously been mapped genetically. Partial sequence analysis verifies the presence of cSOD-coding sequences in this clone and indicates that the intron structure of the Drosophila cSOD gene differs significantly from its human counterpart.     

小麦生长素结合基因 TaABP1-D的定位表达及染色体定位

生长素影响了植物生长发育的诸多过程。生长素结合蛋白 ABP1 (auxin binding protein) 作为一种生长素受体,在质膜上生长素诱导的快速反应中起重要作用。小麦中已经克隆得到了TaABP1-D,但其在细胞中的作用位置以及在染色体定位情况仍不明确。本实验利用洋葱表皮细胞瞬时表达系统对小麦生长素结合基因 TaABP1-D进行亚细胞定位,表明TaABP1-D蛋白为膜蛋白,存在于细胞质和细胞膜中;同时利用中国春缺体-四体材料和信息学方法,将TaABP1-D定位在小麦5D染色体长臂的近着丝粒位置上,距两侧EST标记BE490079和BE405060的遗传距离分别为0.51 cM和0.28 cM。     

Subunits of chromosomal DNA

A study of chromosomal DNA from Chinese hamster cells and chick fibroblasts by electron microscopy after partial denaturation revealed small regions which melted at 50° and could be stabilized by reaction with formaldehyde. The melted regions remained open so that their length and distribution along the DNA strands could be measured. The measurements indicated regularly spaced sites with low melting points at 0.4–0.5 micron intervals in most of the DNA. The length of the melted regions varied from those just visible to some as long as 0.4–0.5 microns, which probably represents the entire region between two successive sites with low melting points. A computer analysis of the spacings indicated a high probability of melted sites occurring every 2 microns along the DNA strands. Both of these spacings correspond to functional subunits of the DNA which can be isolated under appropriate metabolic conditions.