Rearrangements of yeast chromosomes revealed by pulsed field gel electrophoresis

Electrophoretic karyotypes of yeast Saccharomyces cerevisiae integrant strains containing the pYF91 plasmid integrated into the chromosomes I, III, VI, IX, XI were studied. A possibility was demonstrated of visual identification of the chimaeric chromosomes via the molecular weight increase by 13200 bp (the plasmid size) determined by pulsed field gel electrophoresis. Several gamma-rays induced rearrangements of the yeast chimaeric chromosome I causing instability in hybrids were also studied. The deletions induced in the I chromosome were analysed and their size estimated. The technique of pulsed field gel electrophoresis is recommended for determination of insertions and deletions in the chromosomes of yeast Saccharomyces cerevisiae.     

Organization and evolution of an alpha satellite DNA subset shared by human chromosomes 13 and 21

The structure of the alpha satellite DNA higher-order repeat (HOR) unit from a subset shared by human chromosomes 13 and 21 (D13Z1 and D21Z1) has been examined in detail. By using a panel of hybrids possessing either a chromosome 13 or a chromosome 21, different HOR unit genotypes on chromosomes 13 and 21 have been distinguished. We have also determined the basis for a variant HOR unit structure found on 8% of chromosomes 13 but not at all on chromosomes 21. Genomic restriction maps of the HOR units found on the two chromosome 13 genotypes and on the chromosome 21 genotype are constructed and compared. The nucleotide sequence of a predominant 1.9-kilobasepair HOR unit from the D13Z1/D21Z1 subset has been determined. The DNA sequences of different alpha satellite monomers comprising the HOR are compared, and the data are used to develop a model, based on unequal crossing-over, for the evolution of the current HOR unit found at the centromeres of both these chromosomes.Correspondence to: H.F. Willard     

Extensive chromosomal polymorphism revealed by ribosomal DNA and satellite DNA loci in 13 Citrus species

Little is known about the chromosomal variability and polymorphism existing in mitotic chromosomes of Citrus, mainly due to lack of reliable chromosomal markers and small chromosome size. To test the hypothesis of chromosomal polymorphism and provide the foundation of the genome organization in the Citrus cultivars, we have developed molecular cytogenetic markers for 13 Citrus species collected from Jeju island, Korea. In this study, we demonstrated that the chromosomal locations of cytogenetic markers are quite variable and extremely polymorphic, in contrast to the previous studies. The data obtained in this study will be of utmost importance in cytological systematics and karyotyping of the Citrus species.     

Four distinct alpha satellite subfamilies shared by human chromosomes 13, 14 and 21.

We describe the characterisation of four alpha satellite sequences which are found on a subset of the human acrocentric chromosomes. Direct sequence study, and analysis of somatic cell hybrids carrying specific human chromosomes indicate a unique 'higher-order structure' for each of the four sequences, suggesting that they belong to different subfamilies of alpha DNA. Under very high stringency of Southern hybridisation conditions, all four subfamilies were detected on chromosomes 13, 14 and 21, with 13 and 21 showing a slightly greater sequence homology in comparison to chromosome 14. None of these subfamilies were detected on chromosomes 15 and 22. In addition, we report preliminary evidence for a new alphoid subfamily that is specific for human chromosome 14. These results, together with those of earlier published work, indicate that the centromeres of the five acrocentric chromosomes are characterised by a number of clearly defined alphoid subfamilies or microdomains (with at least 5, 7, 3, 5 and 2 different ones on chromosomes 13, 14, 15, 21 and 22, respectively). These microdomains must impose a relatively stringent subregional pairing of the centromeres of two homologous chromosomes. The different alphoid subfamilies reported should serve as useful markers to allow further 'dissection' of the structure of the human centromere as well as the investigation of how the different nonhomologous chromosomes may interact in the aetiology of aberrations involving these chromosomes.     

Flexibility difference between double-stranded RNA and DNA as revealed by gel electrophoresis

A systematic study of agarose gel electrophoresis of double-stranded RNA in the kilobase range of sizes was performed. The dsRNA to dsDNA relative mobility was found to depend on gel concentration: in low density gels RNA moves slower and in high density gels - faster than DNA of the same molecular size. The electrophoretic differences were interpreted within the reptation theory to be mainly due to the molecular stiffness differences. The dsRNA persistence length was roughly estimated to be about twice as great as that of DNA.     

Comparison of Streptococcus thermophilus strains by pulse field gel electrophoresis of genomic DNA

Pulse field gel electrophoresis (PFGE) was utilised to compare the genomes of 16 Streptococcus thermophilus cultures from yoghurt, cheese, laban and dahi after digestion with the restriction endonucleases, SfiI, SmaI and BssHII. PFGE profiles could be used for strain identification and were also useful in predicting relatedness of certain strains. Genetic variations between specific morphotypes of a highly proteolytic culture were not detectable by PFGE in this study. Statistical analysis of SmaI restriction patterns enabled the clustering of strains into two groups which corresponded with biochemical properties of the strains examined and suggested that PFGE profiles could be useful in predicting biochemical characteristics.     

An alpha satellite DNA polymorphism specific for the centromeric region of chromosome 13

Alpha satellite DNA is composed of variants of a short consensus sequence that are repeated in tandem arrays in the centromeric heterochromatin of each human chromosome. To define centromeric markers for linkage studies, we screened human genomic DNA for restriction fragment length polymorphisms using a probe detecting alphoid sequences on chromosomes 13 and 21. We describe one such DNA polymorphism. Analysis of linkage of this DNA marker to other polymorphic markers in the CEPH pedigrees demonstrates linkage to markers on the proximal long arm of chromosome 13 and defines the centromeric end of the linkage map of this chromosome.     

Discrimination between alpha-satellite DNA sequences from chromosomes 21 and 13 by using polymerase chain reaction.

alpha-Satellite subfamilies from chromosomes 21 and 13 are almost identical in sequence and cannot be distinguished from each other by hybridization techniques. A general method based on membrane-bound PCR is described here, allowing the discrimination of alpha-satellite DNA sequences from each of these two chromosomes, after detection by Southern blot hybridization. The PCR conditions were developed using somatic hybrid DNAs. The method was tested in membrane-bound PCR by using the alpha-satellite bands from a Southern blot of a CEPH family. The chromosomal origin of these bands, previously determined by linkage analysis, was confirmed by this method.     

Model and computer simulations of the motion of DNA molecules during pulse field gel electrophoresis

A model is presented for the motion of individual molecules of DNA undergoing pulse field gel electrophoresis (PFGE). The molecule is represented by a chain of charged beads connected by entropic springs, and the gel is represented by a segmented tube surrounding the beads. This model differs from earlier reptation/tube models in that the tube is allowed to leak in certain places and the chain can double over and flow out of the side of the tube in kinks. It is found that these kinks often lead to the formation of U shapes, which are a major source of retardation in PFGE. The results of computer simulations using this model are compared with real DNA experimental results for the following cases: steady field motion as seen in fluorescence microscopy, mobility in steady fields, mobility in transverse field alternation gel electrophoresis (TFAGE), mobility in field inversion gel electrophoresis (FIGE), and linear dichroism (LD) of DNA in agarose gels during PFGE. Good agreement between the simulations and the experimental results is obtained.     

Homologous alpha satellite sequences on human acrocentric chromosomes with selectivity for chromosomes 13, 14 and 21: implications for recombination between nonhomologues and Robertsonian translocations.

We report a new subfamily of alpha satellite DNA (pTRA-2) which is found on all the human acrocentric chromosomes. The alphoid nature of the cloned DNA was established by partial sequencing. Southern analysis of restriction enzyme-digested DNA fragments from mouse/human hybrid cells containing only human chromosome 21 showed that the predominant higher-order repeating unit for pTRA-2 is a 3.9 kb structure. Analysis of a "consensus" in situ hybridisation profile derived from 13 normal individuals revealed the localisation of 73% of all centromeric autoradiographic grains over the five acrocentric chromosomes, with the following distribution: 20.4%, 21.5%, 17.1%, 7.3% and 6.5% on chromosomes 13, 14, 21, 15 and 22 respectively. An average of 1.4% of grains was found on the centromere of each of the remaining 19 nonacrocentric chromosomes. These results indicate the presence of a common subfamily of alpha satellite DNA on the five acrocentric chromosomes and suggest an evolutionary process consistent with recombination exchange of sequences between the nonhomologues. The results further suggests that such exchanges are more selective for chromosomes 13, 14 and 21 than for chromosomes 15 and 22. The possible role of centromeric alpha satellite DNA in the aetiology of 13q14q and 14q21q Robertsonian translocations involving the common and nonrandom association of chromosomes 13 and 14, and 14 and 21 is discussed.     

The pachytene chromosomes of maize as revealed by fluorescence in situ hybridization with repetitive DNA sequences

A repetitive DNA sequence, ZmCR2.6c, was isolated from maize based on centromeric sequence CCS1 of the wild grass Brachypodium sylvaticum. ZmCR2.6c is 309 bp in length and shares 65% homology to bases 421–721 of the sorghum centromeric sequence pSau3A9. Fluorescence in situ hybridization (FISH) localized ZmCR2.6c to the primary constrictions of pachytene bivalents and to the stretched regions of MI/AI chromosomes, indicating that ZmCR2.6c is an important part of the centromere. Based on measurements of chromosome lengths and the positions of FISH signals of several cells, a pachytene karyotype was constructed for maize inbred line KYS. The karyotype agrees well with those derived from traditional analyses. Four classes of tandemly repeated sequences were mapped to the karyotype by FISH. Repeats 180 bp long are present in cytologically detectable knobs on 5L, 6S, 6L, 7L, and 9S, as well as at the termini and in the interstitial regions of many chromosomes not reported previously. A most interesting finding is the presence of 180-bp repeats in the NOR-secondary constriction. TR-1 elements co-exist with 180-bp repeats in the knob on 6S and form alone a small cluster in 4L. 26S and 5S rRNA genes are located in the NOR and at 2L.88, respectively. The combination of chromosome length, centromere position, and distribution of the tandem repeats allows all chromosomes to be identified unambiguously. The results presented form an important basis for using FISH for physical mapping and for investigating genome organization in maize. Received: 29 June 1999 / Accepted: 10 November 1999     

Evolutionary relationships of multiple alpha satellite subfamilies in the centromeres of human chromosomes 13, 14, and 21

Summary Using Southern and in situ hybridization analyses, we have earlier defined four different subfamilies of alpha satellite DNA (designated pTRA-1, -2, -4, and -7), each of which has a unique higher order structure represented almost identically on human chromosomes 13, 14, and 21. Here we present the complete sequence of single isolates of these four subfamilies, representing approximately 12 kb of sequence information. Sequences of the individual 171-bp monomers that constitute these four subfamilies (and a fifth subfamily, Alpha-R1, that is known to be present on chromosomes 13 and 21) were compared both within and between the different clones. The results indicate that, at the level of their primary sequence, the five alpha subfamilies are characterized by structures that are as unrelated to each other as the different alpha subfamilies from other chromosomes. However, sequence comparisons between monomers of these clones indicate the possibility that pTRA-2, -4, and-1 may have arisen, at least in part, from a common ancestral alphoid sequence. We also provide evidence that exchange of pTRA-1 between nonhomologous centromeres and its homogenization throughout the population, perhaps by unequal exchange mechanisms, could have occurred after the divergence of humans and chimpanzees. The evolution of multiple alphoid subfamilies within a single centromere suggests that unequal exchange mechanisms may be restricted to specific domains. This may in turn contribute to some requirement for subregional pairing of sequences along the length of the centromeres of these chromosomes. Offprint requests to: K.H.A. Choo     

Genomic diversity within the genus Pediococcus as revealed by randomly amplified polymorphic DNA PCR and pulsed-field gel electrophoresis

The genomic diversity of 33 previously assigned strains from six species within the genus Pediococcus was assessed by randomly amplified polymorphic DNA (RAPD) PCR and pulsed-field-gel electrophoresis (PFGE). The RAPD PCR patterns produced by two separate random primers, termed P1 (ACGCGCCCT) and P2 (ATGTAACGCC), were compared by the Pearson correlation coefficient and the unweighted pair group method with arithmetic averages clustering algorithm. Pattern variations between repeat samples set a strain discrimination threshold of less than 70% similarity. P1 and P2 primers alone and in combination produced 14, 21, and 28 distinct patterns, respectively. When each strain was assigned with a type strain with which it shared the highest level of similarity, both primers grouped 17 of the 27 strains to their proposed species. PFGE following genomic digestion with the restriction enzymes ApaI, NotI, and AscI produced 30, 32, and 28 distinct macrorestriction patterns, respectively. Specific DNA fragments within the NotI and AscI macrorestriction patterns for each strain were observed that allowed 27 of the 33 strains to be assigned to their proposed species. For example, following digestion with AscI, all Pediococcus parvulus strains were characterized by two DNA fragments, one of approximately 220 kb and another between 700 and 800 kb. The exceptions correlated with those observed with both RAPD PCR primers and included three P. damnosus and two P. pentosaceus strains that grew at temperatures regarded as nonpermissive for their proposed species but not for those with which they grouped.     

Detection of specific sequences among DNA fragments separated by gel electrophoresis.

Ribosomal RNA and precursor ribosomal RNA from at least one representative of each vertebrate class have been analyzed by electron microscopic secondary structure mapping. Reproducible patterns of hairpin loops were found in both 28 S ribosomal and precursor ribosomal RNA, whereas almost all the 18 S ribosomal RNA molecules lack secondary structure under the spreading conditions used. The precursor ribosomal RNA of all species analyzed have a common design. The 28 S ribosomal RNA is located at or near the presumed 5′-end and is separated from the 18 S ribosomal RNA region by the internal spacer region. In addition there is an external spacer region at the 3′-end of all precursor ribosomal RNA molecules. Changes in the length of these spacer regions are mainly responsible for the increase in size of the precursor ribosomal RNA during vertebrate evolution. In cold blooded vertebrates the precursor contains two short spacer regions; in birds the precursor bears a long internal and a short external spacer region, and in mammals it has two long spacer regions. The molecular weights, as determined from the electron micrographs, are 2·6 to 2·8 × 10⁶ for the precursor ribosomal RNA of cold blooded vertebrates, 3·7 to 3·9 × 10⁶ for the precursor of birds, and 4·2 to 4·7 × 10⁶ for the mammalian precursor. Ribosomal RNA and precursor ribosomal RNA of mammals have a higher proportion of secondary structure loops when compared to lower vertebrates. This observation was confirmed by digesting ribosomal RNAs and precursor ribosomal RNAs with single-strandspecific S₁ nuclease in aqueous solution. Analysis of the double-stranded, S₁-resistant fragments indicates that there is a direct relationship between the hairpin loops seen in the electron microscope and secondary structure in aqueous solution.     

Clonal differences within M-types of the group A streptococcus revealed by pulsed field gel electrophoresis

Digestion of chromosomal DNA with the rare cutting restriction enzyme SfiI in association with pulsed field gel electrophoresis was used to observe restriction fragment length polymorphisms (RFLP) among isolates of group A Streptococcus. Streptococci examined included isolates belonging to the same M-type (epidemiologically related and unrelated), and isolates from other M-types. RFLP patterns were quite distinct between all serotypes tested. More importantly, isolates from within a serotype could be differentiated by this technique.     

Characterization and chromosomal organization of satellite DNA sequences in Picea abies

Three clones containing satellite DNA sequences were selected from a randomly sheared genomic DNA library of Picea abies (clones PAF1, PAG004P22F (2F), and PAG004E03C (3C)). PAF1 contained 7 repeats that were 37-55 bp in length and had 68.9%-91.9% nucleotide sequence similarity. Two 2F repeats were 305-306 bp in length and had 83% sequence similarity. Two 3C repeats were 193-226 bp in length and had a sequence similarity of 78.6%. The copy number per 1C DNA of PAF1, 2F, and 3C repeats was 2.7 x 106, 2.9 x 105, and 2.9 x 104, respectively. In situ hybridization showed centromeric localization of these sequences in two chromosome pairs with PAF1, all pairs but one with 2F, and three pairs with 3C. Moreover, PAF1 sequences hybridized at secondary constrictions in six pairs, while 2F-related sequences were found at these chromosome regions only in four pairs. These hybridization patterns allow all chromosome pairs to be distinguished. PAF1-related repeats were contained in the intergenic spacer (IGS) of ribosomal cistrons in all six nucleolar organizers of the complement, while sequences related to 2F were found on only one side of the rDNA arrays in four pairs, showing structural diversity between rDNA regions of different chromosomes.