Primate evolution of a dispersed human repetitive DNA sequence

A dispersed middle repetitive DNA sequence isolated originally from human chromosome 12 did not show homology with rodent DNA under standard conditions of Southern DNA blot analysis. The evolutionary relationship of this human repetitive DNA to that of other primates was investigated using three hybridization methods: DNA dot blot, Southern DNA blot analysis, and chromosome in situ hybridization. Homology with the human repetitive DNA was found throughout the suborder Anthropoidea, in fourteen ape and New and Old World monkey species. In addition, the human pattern of hybridization to noncentromeric regions of all chromosomes was seen. No hybridization by any of the three techniques was found in five species of the suborder Prosimii. The phenomenon of marked differences in sequence homology and copy number of dispersed repetitive DNA from closely related species has been observed in protozoans (Plasmodia), Drosophila, sea urchins, mice and the great apes (Hominoidea). We report here a similar phenomenon that may have occurred at an early stage in primate evolution.     

Characterization of a major polymorphic tandem repeat in Mycobacterium tuberculosis and its potential use in the epidemiology of Mycobacterium kansasii and Mycobacterium gordonae.

In this study, the occurrence of repeated DNA sequences in the chromosome of Mycobacterium tuberculosis was investigated systematically. By screening a M. tuberculosis lambda gt-11 gene library with labeled total chromosomal DNA, five strongly hybridizing recombinants were selected, and these contained DNA sequences that were present in multiple copies in the chromosome of M. tuberculosis. These recombinants all contained repeated sequences belonging to a single family of repetitive DNA, which shares homology with a previously described repeated sequence present in recombinant pPH7301. Sequences analysis of pPH7301 showed the presence of a 10-bp sequence that was tandemly repeated and invariably separated by 5-bp unique spacer sequences. Southern blot analysis revealed that the majority of the repeated DNA in M. tuberculosis is composed of this family of repetitive DNA. Because the 10-bp repeats are slightly heterogeneous in sequence, we designated this DNA as a major polymorphic tandem repeat, MPTR. The presence of this repeated sequence in various other mycobacterial species was investigated. Among the MPTR-containing mycobacterial species the chromosomal location of the repetitive DNA is highly variable. The potential use of this polymorphism in the epidemiology of mycobacterioses is discussed.     

Studies on DNA sequences in the Osmundaceae

Summary Phylogenetic relationships ofOsmunda cinnamomea, O. claytoniana, andO. regalis were explored by means of DNA sequence comparisons. Hydroxyapatite thermal elution profiles of self-reassociated repetitive DNA fragments were very similar, indicating the absence of gross differences in the amount of recent amplification or addition of repetitive DNA in any of these three genomes. Interspecific DNA sequence comparisons showed, in contrast to our earlier interpretation, that repeated DNA sequences ofO. claytoniana are nearly equally diverged from those ofO. cinnamomea andO. regalis. Differences between repetitive sequences of the three species can be interpreted as reflecting amplification events which occurred subsequent to speciation. The data obtained suggest that the threeOsmunda species most likely arose more or less simultaneously from a common ancestor. These findings were verified in experiments with tracer DNA preparations enriched for single copy sequences. On the basis of the hybridization data presented here and of the fossil record, the rate of single copy sequence divergence in the ferns is comparable to that in the primates, although slower than that observed in other animal taxa. From this first evaluation of rates of DNA evolution in plants it would seem that the rates for plants and animals are roughly comparable. The evidence suggests that species divergence is accompanied by further reiteration of preexisting repeat sequences. The rate of addition of repetitive sequences probably is slower in ferns than in angiosperms. This difference might be attributable to the much larger effective generation time in ferns.     

Isolation, localization, and physical mapping of a highly polymorphic locus on human chromosome 11q13.

A highly polymorphic repetitive sequence, D11S533, was isolated by oligonucleotide hybridization from an arrayed chromosome 11q-specific cosmid library. The DNA sequence of this element was determined and found to consist of a repetitive degenerate hexanucleotide sequence [T(Pu)T(Pu)T(Pu)]n extending over 438 bp. Southern blot analysis demonstrated that this element is relatively unique in the human genome. This sequence can be detected by amplification using the polymerase chain reaction (PCR) with oligonucleotide primers complementary to unique sequences flanking the repetitive element. This sequence displays a high degree of polymorphism, and analysis of 15 individuals demonstrated at least 10 alleles ranging in size from 300 to 900 bp. Fluorescence in situ hybridization was used to localize this sequence to 11q13 (FLpter 0.60 +/- 0.02). Pulsed-field gel electrophoresis and the isolation of yeast artificial chromosomes established the long-range physical map surrounding the locus. Because various alleles of this polymorphic sequence can be easily detected by PCR amplification, this probe has potential usefulness in genetic linkage mapping as well as identity testing.     

Molecular and cytological characterization of repetitive DNA sequences in Brassica

Summary We isolated three different repetitive DNA sequences from B. campestris and determined their nucleotide sequences. In order to analyze organization of these repetitive sequences in Brassica, Southern blot hybridization and in situ hybridization with metaphase chromosomes were performed. The sequence cloned in the plasmid pCS1 represented a middle repetitive sequence present only in B. campestris and not detected in closely related B. Oleracea. This sequence was localized at centromeric regions of six specific chromosomes of B. campestris. The second plasmid, pBT4, contained a part of the 25S ribosomal RNA gene, and its copy number was estimated to be 1,590 and 1,300 per haploid genome for B. campestris and B. oleracea, respectively. In situ hybridization with this sequence showed a clear signal at the NOR region found in the second largest chromosome of B. Campestris. The third plasmid, pBT11, contained a 175-bp insert that belongs to a major family of tandem repeats found in all the Brassica species. This sequence was detected at centromeric regions of all the B. campestris chromosomes. Our study indicates that in situ hybridization with various types of repetitive sequences should give important information on the evolution of repetitive DNA in Brassica species.     

Effect of a recA mutation on cholera toxin gene amplification and deletion events.

The cholera toxin operon (ctxAB) is located on a 7-kilobase pair variable genetic element which undergoes genetic duplication and amplification events in Vibrio cholerae. Amplification of the ctx genetic element was investigated by substituting the resident ctx loci of two V. cholerae strains with a DNA fragment encoding resistance to kanamycin. Although these strains were not normally resistant to greater than 150 micrograms of kanamycin per ml, spontaneous derivatives could be obtained that grew well on 3 mg of kanamycin per ml. Southern blot analysis of these highly resistant isolates demonstrated that the ctx element was amplified approximately 20-fold. This amplification process was completely inhibited in the absence of a functional recA gene. The V. cholerae RecA protein, therefore, is essential for cholera toxin gene amplification. Spontaneous deletions of the ctx structural genes were observed in both recA+ and recA- V. cholerae strains, although such deletions occurred at a 21-fold-lower frequency in the latter case. Structural analysis of these ctx amplification and deletion events supports a model for their formation that involves unequal crossing over between repetitive sequences located upstream and downstream of the ctx operon.     

Studies of a novel repetitive sequence family in the genome of mice

A new middle repetitive sequence is described in the mouse genome. It has been revealed with a recombinant clone isolated from a Mus musculus BamHI gene library constructed in pBR322 and containing an insertion of 1.73 kb. When digests of genomic DNA were subjected to Southern blot hybridization, using the 1.73-kb insert as probe, we obtained a light smear and discrete bands, indicating a dispersion in the mouse genome of this sequence. This 1.73-kb sequence seems to be a part of a greater repetitive sequence at least 6 kb in length. The sizes of the bands hybridizing with the 1.73-kb insert are similar when compared between different laboratory strains but differ remarkably between the two species M. musculus and Mus caroli. We have shown also a great variation in the copy number of the sequence studied between these two species. When rat DNA is probed with the 1.73-kb insert, no hybridization is observed. Subcloning of the 1.73-kb sequence in three fragments has pointed out that the reiteration was not homogeneous along the 1.73-kb sequence. The 1.73-kb clone was sequenced and compared with other interspersed repetitive sequences, previously described in the rodent genome, and no homology was found.     

Phylogeny of ruminants secretory ribonuclease gene sequences of pronghorn (Antilocapra americana)

Phylogenetic analyses based on primary structures of mammalian ribonucleases, indicated that three homologous enzymes (pancreatic, seminal and brain ribonucleases) present in the bovine species are the results of gene duplication events, which occurred in the ancestor of the ruminants after divergence from other artiodactyls. In this paper sequences are presented of genes encoding pancreatic and brain-type ribonuclease genes of pronghorn (Antilocapra americana). The seminal-type ribonuclease gene could not be detected in this species, neither by PCR amplification nor by Southern blot analyses, indicating that it may be deleted completely in this species. Previously we demonstrated of a study of amino acid sequences of pancreatic ribonucleases of a large number of ruminants the monophyly of bovids and cervids, and that pronghorn groups with giraffe. Here we present phylogenetic analyses of nucleotide sequences of ribonucleases and other molecules from ruminant species and compare these with published data. Chevrotain (Tragulus) always groups with the other ruminants as separate taxon from the pecora or true ruminants. Within the pecora the relationships between Bovidae, Cervidae, Giraffidae, and pronghorn (Antilocapra) cannot be decided with certainty, although in the majority of analyses Antilocapra diverges first, separately or joined with giraffe. Broad taxon sampling and investigation of specific sequence features may be as important for reliable conclusions in phylogeny as the lengths of analyzed sequences.     

The genomic organization and evolutionary distribution of a tandemly repeated DNA sequence family in the genus Crocus (Iridaceae)

From a recombinant DNA-library from Crocus vernus, two closely related clones of highly repetitive DNA, pCvKB7 and pCvKB8, were sequenced and their genomic distribution and organization were investigated by Southern and in situ hybridization. The lengths of the clones were 181 and 178 bp respectively; the sequences were approximately 85% identical, and thus belonged to a sequence family, named the pCvKB8-family. No homologous sequences were found in the databases (BLAST made may 2004). The presence of pCvKB8 in 52 Crocus species and six species from other genera were analyzed by Southern hybridization. The sequence family was essentially Crocus-specific. However, the distribution of hybridization signal across the genus showed poor agreement with the taxonomic structure of the Crocus genus, suggesting that the subdivision does not follow the phylogeny of this sequence family. The chromosomal distribution on three Crocus species was essentially identical: tandem organization close to all telomeres and most centromeres, with a few additional intercalary sites.     

Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes.

Dispersed repetitive DNA sequences have been described recently in eubacteria. To assess the distribution and evolutionary conservation of two distinct prokaryotic repetitive elements, consensus oligonucleotides were used in polymerase chain reaction [PCR] amplification and slot blot hybridization experiments with genomic DNA from diverse eubacterial species. Oligonucleotides matching Repetitive Extragenic Palindromic [REP] elements and Enterobacterial Repetitive Intergenic Consensus [ERIC] sequences were synthesized and tested as opposing PCR primers in the amplification of eubacterial genomic DNA. REP and ERIC consensus oligonucleotides produced clearly resolvable bands by agarose gel electrophoresis following PCR amplification. These band patterns provided unambiguous DNA fingerprints of different eubacterial species and strains. Both REP and ERIC probes hybridized preferentially to genomic DNA from Gram-negative enteric bacteria and related species. Widespread distribution of these repetitive DNA elements in the genomes of various microorganisms should enable rapid identification of bacterial species and strains, and be useful for the analysis of prokaryotic genomes.     

A comparative study of the heterochromatin of Apodemus sylvaticus and Apodemus flavicollis

The two closely related species Apodemus sylvaticus and Apodemus flavicollis (Muridae) differ in the distribution of their heterochromatin. Two major repetitive sequences known to occur in both species were isolated from A. flavicollis after digestion of total nuclear DNA with the restriction enzymes HindIII and EcoRI respectively and characterized in both species by filter hybridisation and in situ hybridisation to metaphase chromosomes. The EcoRI clone detects a dispersed repetitive sequence family in the genome of both species. Southern blot hybridisation with the HindIII satellite DNA probe reveals major similarities and minor differences in the two species. In situ hybridisation with the HindIII probe labels all chromosomes of A. flavicollis exclusively in the centromeric heterochromatin, whereas in A. sylvaticus several autosomes are also labelled distally. The labelling patterns correspond to the distribution of heterochromatin in the two species. It is concluded that the additional distal heterochromatin of A. sylvaticus contains similar sequences to those of the centromeric heterochromatin of both species. The distal heterochromatin in A. sylvaticus most likely evolved by transposition and amplification of centromeric satellite DNA elements, after the separation of the two species.     

Repeated DNA sequences in the microbat species Miniopterus schreibersi (Vespertilionidae; Chiroptera)

Repetitive DNA sequences represent a substantial component of eukaryotic genomes. These sequences have been described and characterized in many mammalian species. However, little information about repetitive DNA sequences is available in bat species. Here we describe an EcoRI family of repetitive DNA sequences present in the species Miniopterus schreibersi. These repetitive sequences are 57.85%, A-T rich, organized in tandem, and with a monomer unit length of 904 bp. Methylation analysis using the isoesquizomer pair MspI and HpaII indicates that the cytosines present in the sequences CCGG are partially methylated. Furthermore, Southern blot analysis demonstrated that these DNA sequences are absent in the genomes of four related microbat species and suggest that it could be specific to the M. schreibersi genome.     

The Balbiani ring 2 gene in Chironomus tentans is built from two types of tandemly arranged major repeat units with a common evolutionary origin

The internal structure of the 37 kb long Balbiani ring 2 (BR 2) gene in Chironomus tentans has been studied by analysis of a collection of cloned cDNA sequences and in genomic Southern blot analysis with the cDNA sequences used as probes. The BR 2 gene contains two types of tandemly arranged major repeat units ˜200 bp long, represented in our study by the pCt 7 and the pCt 63 cDNA inserts. The pCt 7 major repeat units are arranged in one or possibly a few blocks and cover ˜10 kb of the gene; the pCt 63 units form one uninterrupted block, 22 kb in length. Genomic Southern blot hybridizations revealed a number of sequence variants of the pCt 7 major repeat unit. In contrast, the ˜100 copies of the pCt 63 major repeat unit seem to be almost identical. The pCt 7 major repeat unit, 180 bp in length, is organized in the same way as the previously described 215 bp long pCt 63 major repeat, i.e., it contains a repetitive and a non-repetitive part. Moreover, the two major repeat units show a high degree of sequence homology, indicating that the pCt 7 and pCt 63 sequence blocks within the Br 2 gene have evolved through stepwise amplification from a common ancestral sequence.     

Characterization of a Tc1-like transposon in the Antarctic ice-fish,Chionodraco hamatus

We report the presence of Tc1 transposon-like sequences in the Antarctic ice-fish Chionodraco hamatus, belonging to the Notothenioidei. The complete DNA sequence of these transposon-like elements is reduced in length compared to other Tc1 transposons, but it appears to share significant structural similarities with them. It contains a degenerate open reading frame, whose inferred 264 amino acid sequence shares sequence similarity with the 'aspartic acid, aspartic acid (35) glutamic acid' family of transposases, particularly those from Caenorhabditis species (sp.) and Drosophila sp. Southern blot analysis and polymerase chain reaction amplification indicate that Tc1 transposon-like sequences are present in other notothenioid species, though their amount can vary in the different lineages.     

Origin, dispersal and genomic structure of a low-copy-number hypervariable RFLP clone in Triticum and Aegilops species

The genome of common wheat has evolved through allopolyploidization of three ancestral diploid genomes. A previously identified restriction fragment length polymorphism (RFLP) marker, pTag546, has the unique feature of showing hypervariability among closely related common wheat cultivars. To understand the origin and the mode of dispersal of this hypervariable sequence in the wheat genome, the distribution and structure of the homologous sequences were studied using ancestral diploid species, tetraploid disomic substitution lines and synthetic hexaploid lines. Comparative Southern blot and PCR analyses suggested that pTag546 homologs in the tetraploid and hexaploid wheat were derived from the S genome of Aegilops speltoides. Some pTag546 homologs were found to have transposed to A and D genomes in polyploid wheat. Evidence of transposition and elimination in some synthetic hexaploid lines was also obtained by comparing their copy numbers with those in the parental lines. Southern blot analysis of a genomic clone using a contiguous subset of sequences as probes revealed a core region of hypervariability that coincided with the region containing pTag546. No obvious structural characteristics that could explain the hypervariability, however, were found around the pTag546 sequence, except for accumulation of small repetitive sequences at one border. It was concluded that pTag546 increased its copy number through yet unknown mechanism(s) of transposition to various chromosomal locations over the period of allopolyploid evolution and during the artificial genome manipulation in wheat.     

Short, interspersed, and repetitive DNA sequences in Spiroplasma species

Small fragments of DNA from an 8-kbp plasmid, pRA1, from a plant pathogenic strain of Spiroplasma citri were shown previously to be present in the chromosomal DNA of at least two species of Spiroplasma. We describe here the shot-gun cloning of chromosomal DNA from S. citri Maroc and the identification of two distinct sequences exhibiting homology to pRA1. Further subcloning experiments provided specific molecular probes for the identification of these two sequences in chromosomal DNA from three distinct plant pathogenic species of Spiroplasma. The results of Southern blot hybridization indicated that each of the pRA1-associated sequences is present as multiple copies in short, dispersed, and repetitive sequences in the chromosomes of these three strains. None of the sequences was detectable in chromosomal DNA from an additional nine Spiroplasma strains examined.     

Characterization of a new repetitive sequence in the Dictyostelium discoideum genome

A repetitive DNA sequence was isolated from a Dictyostelium discoideum genomic plasmid library of BglII-digested DNA ligated to the BamHI site in pBR322. This clone, called pBS582, hybridized to a large number of phage lambda Dictyostelium genomic clones. Southern blot analysis indicated that pBS582 DNA hybridized to many differently sized genomic DNA fragments generated by digestion with Eco RI, AvaI, or HindIII. Restriction maps of pBS582 and five genomic clones showed that the flanking regions of each of the genomic clones were different. These findings indicate that the sequence specific to pBS582 is scattered throughout the Dictyostelium genome and is reiterated approximately 100 times in the haploid genome. Northern blot analysis revealed that RNA which hybridized to pBS582 DNA was present during all stages of growth and development and did not seem to be developmentally regulated. Southern blot analysis of DNAs from other slime molds (D. giganteum, D. purpureum, and Polysphondylium violaceum) were performed to determine whether the pBS582 sequence was present in other species of slime molds. Hybridization of pBS582 was observed to DNA from the two Dictyostelium species but not to Polysphondylium. It may thus be possible to use hybridization of specific sequences as a biochemical tool to study the relatedness of different slime mold species and their molecular taxonomy.     

Identification of mRNA that encodes an alternative form of H-CAM(CD44) in lymphoid and nonlymphoid tissues

The cluster of differentiation 44 (CD44), hereafter referred to as H-CAM(CD44), represents a novel class of polymorphic (M _r 80 000–215 000) cell adhesion molecules that are involved in cell-cell and cell-matrix adhesion events in a variety of organ systems. We report the detection of distinct mRNAs, in both hematopoietic and nonhematopoietic human cell lines, that encode H-CAM(CD44) with different cytoplasmic domains. Genomic Southern blot analyses indicate that the exons encoding these two cytoplasmic domains are located on the same 16 kilobase (kb) Eco RI restriction fragment. Restriction endonuclease and Southern blot analyses performed on polymerase chain reaction (PCR) amplification copies of these mRNAs confirm that their sequences correspond with previously reported cDNA sequences. A consensus splice donor site which is conserved in human, baboon, and mouse mRNAs that encode a molecule with an elongated cytoplasmic domain (H-CAM-L) is utilized to generate a distinct but low-abundance mRNA species that encodes H-CAM(CD44) with a truncated cytoplasmic domain of only three amino acids (H-CAM-S). Estimations of the relative abundance of these mRNA species in B-lymphoblastoid cells using the PCR amplification technique exhibit average H-CAM-L/H-CAM-S ratios ranging between 100 and 200. Therefore, H-CAM(CD44)-mediated adhesive events may be regulated through a differential capacity of H-CAM-L and H-CAM-S to interact with the cytoskeleton and to participate in intracellular signaling events.     

Cloning and characterization of repetitive DNA sequences from genomes of Oryza minuta and Oryza australiensis

The value of genome-specific repetitive DNA sequences for use as molecular markers in studying genome differentiation was investigated. Five repetitive DNA sequences from wild species of rice were cloned. Four of the clones, pOm1, pOm4, pOmA536, and pOmPB10, were isolated from Oryza minuta accession 101141 (BBCC genomes), and one clone, pOa237, was isolated from Oryza australiensis accession 100882 (EE genome). Southern blot hybridization to different rice genomes showed strong hybridization of all five clones to O. minuta genomic DNA and no cross hybridization to genomic DNA from Oryza sativa (AA genome). The pOm1 and pOmA536 sequences showed cross hybridization only to all of the wild rice species containing the C genome. However, the pOm4, pOmPB10, and pOa237 sequences showed cross hybridization to O. australiensis genomic DNA in addition to showing hybridization to the O. minuta genomic DNA.