Cloning, characterization and physical mapping of three cervid satellite DNA families in the genome of the Formosan muntjac (Muntiacus reevesi micrurus)

Due to their high sequence diversity even among closely related species, satellite DNA sequences can be a useful molecular marker for phylogenetic and taxonomic analyses. To characterize the satellite DNA in the genome of a native muntjac species of Taiwan, the Formosan muntjac, satellite DNA clones representing three different cervid satellite DNA families from this species were isolated and analyzed. Genomic organization study of these satellite DNAs was also undertaken. Three Formosan muntjac satellite DNA clones were obtained and designated as FM-satI (1,391 bp), FM-satII (1,143 bp) and FM-satIV (1,103 bp), and found to share approximately 82, 81 and 98% sequence homology with the Chinese muntjac satellite I clone (C5), Indian muntjac satellite II clone (Mmv-0.7) and Chinese muntjac satellite IV clone (MR-1.0), respectively. These three satellite DNA families are organized in a pter<--FM-satII-FM-satIV-FM-satI-->qter orientation in the centromeric region with satII closely associated with the telomeric sequences. Satellite DNA sequence comparison, in combination with chromosome data concludes that the Formosan muntjac is likely a subspecies of M. reevesi, closely related to the Chinese muntjac. With the kinetochore satellite II DNA co-localizing with the telomeric sequences, the Formosan muntjac chromosomes could be truly telocentric.     

Molecular analysis of a novel tandemly organized repetitive DNA sequence in<Emphasis Type="Italic">Citrus limon</Emphasis> (L.) Burm

Repetitive sequences constitute a significant component of most eukaryotic genomes, and the isolation and characterization of repetitive DNA sequences provide an insight into the organization of the genome of interest. Here, we report the isolation and molecular analysis of a novel tandemly organized repetitive DNA sequence from the genome of Citrus limon. Digestion of C. limon DNA with Hinf I produced a prominent fragment of approximately 300 bp. Southern blotting revealed a ladder composed of DNA fragments that were multimers of the 300-bp Hinf I band. Thus, Hinf I digestion revealed a novel satellite, which we have called the C. limon satellite DNA 300 (CL300). Sequence analysis shows significant homology between a portion of the CL300 monomer and the transposase box of an En/Spm-like element. The CL300 satellite was also detected in grapefruit, sour orange, trifoliate orange and kumquat. These results suggest that the CL300 repeat is an ancient satellite, and we propose that a significant portion originated by amplification of a genomic region containing the En/Spm-like transposase element.     

Nucleotide sequence of the maize transposable element Mul

A cloned DNA fragment from the maize allele Adhl-S3034 contains all of Mul, an insertion element involved in Robertson's Mutator activity. The element is 1367 base pairs (bp) long and is flanked by nine bp direct repeats of insertion site DNA. It has inverted terminal repeats of 215 and 213 bp showing 95% homology. Within the element are two direct repeats of 104 bp showing 96% homology. Four open reading frames (ORFs) were found, two in each DNA strand. Mul can be divided into two halves, each containing one terminal inverted repeat, an internal direct repeat, and two overlapping ORFs. The GC content of each half is high (70%), while that of a central 60 base portion of the element is low (26%). The central region contains the only sequence resembling the TAATA Goldberg and Hogness eukaryotic promoter signal. Multiple copies of DNA sequences related to Mul found in Mutator maize plants are generally similar in organization to the cloned element. A larger version containing a discrete 300 to 400 base pair insertion was found in some Mutator lines.     

Sequence organization and cytological localization of the minor satellite of mouse.

A complete 120 bp genomic consensus sequence for the mouse minor satellite has been determined from enriched L929 centromeric sequences. The extensive sequence homology existing between the major and minor satellite suggests an evolutionary relationship. Some sequences flanking the minor satellite has also been identified and they provide insight into centromeric DNA organization. Isotopic in situ hybridization analysis of the minor satellite to mouse L929 and Mus musculus metaphase spreads showed that this repetitive DNA class is localized specifically to centromeres of all chromosomes of the karyotype. With the use of high resolution non-isotopic fluorescence in situ hybridization the minor satellite is further localized to the outer surface of the centromere in a discrete region at or immediately adjacent to the kinetochore. Our cytological data suggests that the minor satellite might play a role in the organization of the kinetochore region rather than, as previously suggested, sites for general anchoring of the genome to the nuclear matrix.     

Analysis of the subtelomeric regions of macronuclear gene-sized DNA molecules of the hypotrichous ciliate Stylonychia lemnae: Implications for the DNA fragmentation process during macronuclear development?

The subtelomeric regions of macronuclear gene-sized DNA molecules from Stylonychia lemnae were analyzed. The results obtained indicate that these regions show a highly ordered and common sequence organization: Immediately adjacent to the telomeric sequence a short inverted repeat sequence is found, followed by another 7–9 bp inverted repeat sequence at approximately position 40. A 10 bp consensus sequence found in the subtelomeric regions of all gene-sized DNA molecules is found at approximately position 60 and in addition at about the same position palindromic sequences showing no homology to each other are localized. The biological significance of this sequence organization is discussed. © 1993Wiley-Liss, Inc.     

A novel repetitive DNA sequence in lemon [Citrus limon (L.) Burm.] and related species Bruna

Repetitive DNA sequences constitute a significant component of most eukaryotic genomes, and the isolation and characterization of such sequences provide an insight into the organization of the genome of interest. Here, we report the isolation and molecular characterization of a novel repetitive DNA sequence from the genome of Citrus limon. Digestion of C. limon DNA with MboI produced a prominent fragment of approximately 600 bp. Southern blotting revealed a ladder composed of DNA fragments that are multimers of the 600 bp Mbo I band. This suggests that MboI isolated a novel satellite, named C. limon satellite DNA 600 (CL600). Methylation analyses using Sau3AI-MboI isoschizomers demonstrated that most cytosine residues in the GATC sites of this element were methylated in C. limon. This sequence was also found in related citrus plants, like grapefruit and orange, and the hardiest close relatives of Citrus, such as kumquat and trifoliate orange.     

Insertion of the Drosophila transposable element copia generates a 5 base pair duplication

To examine the details of insertion for the D. malanogaster transposable element copia, we have isolated three independent pairs of genomic fragments which correspond to occupied and unoccupied target sites for insertion. Restriction endonuclease analysis suggests that sites with and without an element differ by a simple 5000 bp insertion. Direct DNA sequence analysis demonstrates that a 5 bp sequence, present once in the target DNA at the site of insertion, is found on both sides of the element after insertion. The 5 bp sequences which are duplicated are different in each case. Moreover, there does not appear to be any sequence homology among these three independent insertion sites     

A micronucleus-limited sequence family in Tetrahymena thermophila: organization and sequence conservation.

During macronuclear development in the ciliated protozoan Tetrahymena thermophila, sequence reorganization including sequence loss occurs. Addressing questions about the organization and nucleotide sequence of micronucleus limited regions can lead to insights about mechanisms of DNA rearrangements during macronuclear development as well as mechanisms for the maintenance of the stability of micronucleus-limited sequence families. We have previously identified a moderately repetitive micronucleus-limited sequence family called X-H (family members hybridize to an approximately 450 bp Xbal-HindIII restriction fragment), completely absent from macronuclear DNA. The first member of this family which we isolated is associated with terminal sequences characteristic of a Tel-1 element, a putative micronuclear transposable element. Two additional family members have been isolated which are not closely associated with Tel-1 terminal sequences. We have nucleotide sequence data for three cloned members of the X-H family. This analysis has demonstrated that the longest cloned members of the X-H family share a region of homology of approximately 2,400 bp and are highly conserved, differing only by small insertions or deletions of 100 bp or less. The sequences from one of the sequenced family members flanking the region of homology are themselves mostly micronucleus-limited.     

A micronucleus-limited sequence family in Tetrahymena thermophila: Organization and sequence conservation

During mocronuclear development in the ciliated protozoan Tetrahymena thermophila, sequence reorganization including sequence loss occurs. Addressing questions about the organization and nucleotide sequence of micronucleus limited regions can lead to insights about mechanisms of DNA rearrangements during macronuclear development as well as mechanisms for the maintenance of the stability of micronucleus-limited sequence families. We have previously identified a moderately repetitive micronu-cleus-limited sequence family called X-H (family members hybridize to an approximately 450 bp Xbal-HindIII restriction fragment), completely absent from macronuclear DNA. The first member of this family which we isolated is associated with terminal sequences characteristic of a Tel-1 element, a putative micronuclear transposable element. Two additional family members have been isolated which are not closely associated with Tel-1 terminal sequences. We have nucleotide sequence data for three cloned members of the X-H family. This analysis has demonstrated that the longest cloned members of the X-H family share a region of homology of approximately 2,400 bp and are highly conserved, differing only by small insertions or deletions of 100 bp or less. The sequences from one of the sequenced family members flanking the region of homology are themselves mostly micronucleus-limited. © 1992 Wiley-Liss, Inc.     

Apparent relatedness of the main component of ovine 1.714 satellite DNA to bovine 1.715 satellite DNA

The nucleotide sequence of the principal component of ovine 1.714 g/cm3 satellite DNA was determined from a monomeric fragment inserted at the BamHI site of pBR322 and cloned in Escherichia coli strain RR1. The 816-bp tandemly repeated sequence contains a number of small repeated sequences dispersed within it, one group of which forms a pentameric tandem repeat of a 13-bp segment (positions 548-612). A 20-bp region (60-79) shows an 85% homology with the reverse-complement of the sequence from 455 through 474. There are two regions of 67 bp (75-141) and 59 bp (755-813) which show greater than 70% homology with regions of bovine 1.715 g/cm3 satellite DNA (1402 bp; positions 1218-1284 and 1079-1137, respectively) while a 31-bp region (ovine 62-92, bovine 133-163) shows 80% homology. Quasi-correlation coefficients (Qr) were determined using the triplet numbers of the sheep satellite versus all sequences in the National Biomedical Research Foundation and EMBL nucleotide sequence data bases. Qr equals 0.85 for ovine 1.714 g/cm3 satellite versus bovine 1.715 g/cm3 satellite. The next highest Qr for a bovine satellite segment was 0.58. Thus, the ovine 1.714 g/cm3 and bovine 1.715 g/cm3 satellite appear demonstrably related. Taking into account that sheep and cattle diverged 18-20 million years ago, this suggests that the material may be functional and that its function is related to its sequence.     

Detection of Trypanosoma cruzi DNA within murine cardiac tissue sections by in situ polymerase chain reaction

The use of in situ techniques to detect DNA and RNA sequences has proven to be an invaluable technique with paraffin-embedded tissue. Advances in non-radioactive detection systems have further made these procedures shorter and safer. We report the detection of Trypanosoma cruzi, the causative agent of Chagas disease, via indirect and direct in situ polymerace chain reaction within paraffin-embedded murine cardiac tissue sections. The presence of three T. cruzi specific DNA sequences were evaluated: a 122 base pair (bp) sequence localized within the minicircle network, a 188 bp satellite nuclear repetitive sequence and a 177 bp sequence that codes for a flagellar protein. In situ hybridization alone was sensitive enough to detect all three T. cruzi specific DNA sequences.     

The 724 family of DNA sequences is interspersed about the pericentromeric regions of human acrocentric chromosomes

We describe the organization of the complex, interspersed 724 family of DNA sequences that is distributed in multiple copies about the pericentromeric region of human acrocentric chromosomes. 724 family members were isolated using an efficient recombination-based assay for nucleotide sequence homology to screen a human genomic library. Eight related but distinct 724 family members were isolated that hybridized to a total of 20 different human-genomic EcoRI DNA fragments spanning 100,000 base pairs. In contrast with tandemly clustered satellite and ribosomal DNA sequences also located on the short arms of human acrocentric chromosomes, 724 family members are interspersed. No evidence for local interspersion or homology between 724 family members and ribosomal or satellite DNA sequences was found. Juxtaposition of the complex 724 family to the nucleolus organizer region was a recent event in primate evolution. The unique organization of 724 family members on each of the five human acrocentric chromosomes indicates that the 724 family continues to evolve within the human karyotype.     

Characterization and comparative sequence analysis of replication origins from three large Bacillus thuringiensis plasmids.

The replication origins of three large Bacillus thuringiensis plasmids, derived from B. thuringiensis HD263 subsp. kurstaki, have been cloned in Escherichia coli and sequenced. The replication origins, designated ori 43, ori 44, and ori 60, were isolated from plasmids of 43, 44, and 60 MDa, respectively. Each cloned replication origin exhibits incompatibility with the resident B. thuringiensis plasmid from which it was derived. Recombinant plasmids containing the three replication origins varied in their ability to transform strains of B. thuringiensis, Bacillus megaterium, and Bacillus subtilis. Analysis of the derived nucleotide and amino acid sequences indicates that the replication origins are nonhomologous, implying independent derivations. No significant homology was found to published sequences of replication origins derived from the single-stranded DNA plasmids of gram-positive bacteria, and shuttle vectors containing the three replication origins do not appear to generate single-stranded DNA intermediates in B. thuringiensis. The replication origin regions of the large plasmids are each characterized by a single open reading frame whose product is essential for replication in B. thuringiensis. The putative replication protein of ori 60 exhibits partial homology to the RepA protein of the Bacillus stearothermophilus plasmid pTB19. The putative replication protein of ori 43 exhibits weak but extensive homology to the replication proteins of several streptococcal plasmids, including the open reading frame E replication protein of the conjugative plasmid pAM beta 1. The nucleotide sequence of ori 44 and the amino acid sequence of its putative replication protein appear to be nonhomologous to other published replication origin sequences.     

Nucleotide sequence and functional properties of DNA encoding incompatibility in the broad host-range plasmid R1162

Summary A 370 base pair (bp) fragment of R1162 DNA encoding the incompatibility determinant has been cloned and sequenced. The DNA is located between 6.1 and 6.5 on the R1162 map, near the origin of replication. The sequence contains three perfectly conserved 20 bp direct repeats, with 11 bp of this sequence repeated a fourth time. The direct repeat unit shows some homology with that of another, unrelated broad host-range plasmid, RK2. The cloned DNA has two other properties: it lowers the copy number of R1162 when cloned into this plasmid, and it is required in cis for replication of R1162 satellite plasmids.     

Transfer RNA genes frequently serve as integration sites for prokaryotic genetic elements.

The DNA sequences were determined at the boundaries of the integrated copy of the archaebacterial genetic element SSV1. A 44 bp sequence present as a single copy on the 15.5 kb circular SSV1 DNA flanked the integrated copy as a direct DNA sequence repeat, suggesting that SSV1 integration occurred by recombination between this 44 bp SSV1 sequence and an identical sequence on the bacterial chromosome. At the left attachment site, a region encompassing the 44 bp attachment core sequence and the 31 nucleotides upstream of it displayed all characteristics expected for an arginine tRNA gene. An analysis of published attachment site sequences of other systems revealed that tRNA genes also constitute the bacterial attachment site in the case of three temperate phages and two transmissible plasmids in eubacteria, indicating a widespread occurrence of tRNA genes as integration target sites. This finding may be important for the understanding of mechanisms and evolution of site-specific recombination.     

The invertible P-DNA segment in the chromosome of Escherichia coli.

In the chromosome of many strains of Escherichia coli K12 the excisable element e14 is found, which contains an invertible DNA region. This invertible P region, and the gene responsible for the inversion (pin) were cloned, together with other e14 sequences. The element e14 contains a gene which kills the host cell. This can be repressed by a function also coded by e14. The kil and repressor genes as well as the attachment site of the element were mapped in different regions of the element. The invertible segment and pin gene were sequenced. The invertible segment is 1794 bp long, and contains one large internal open reading frame of 879 bp and reading frames which overlap the end pont of the invertible segment. Although pin highly homologous to gin of phage Mu, neither the genetic organization of the P segment nor the sequence of the putative proteins resemble the invertible G segment of phage Mu (which codes for genes involved in tail fiber assembly). The complete DNA sequences of both invertible segments were screened for homology. No resemblance was found. The P segment is flanked by inverted repeat sequences of 16 bp. Comparison of these with related inversion systems points out that the recombination site maps probably within a 2-bp region. This cross-over site is contained within a short palindromic sequence (AAACC AA GGTTT) which is more or less conserved in the recombination sites of all related DNA invertases.     

Chromosome-specific alpha satellite DNA: nucleotide sequence analysis of the 2.0 kilobasepair repeat from the human X chromosome.

The pericentromeric region of the human X chromosome is characterized by a tandemly repeated family of 2.0 kilobasepair (kb) DNA fragments, initially revealed by cleavage of human DNA with the restriction enzyme BamHI. We report here the complete nucleotide sequence of a cloned member of the repeat family and establish that this X-linked DNA family consists entirely of alpha satellite DNA. Our data indicate that the 2.0 kb repeat consists of twelve alpha satellite monomers arranged in imperfect, direct repeats. Each of the alpha X monomers is approximately 171 basepairs (bp) in length and is 60-75% identical in sequence to previously described primate alpha satellite DNAs. The twelve alpha X monomers are 65-85% identical in sequence to each other and are organized as two adjacent, related blocks of five monomers, plus an additional two monomers also related to monomers within the pentamer blocks. Partial nucleotide sequence of a second, independent copy of the 2.0 kb BamHI fragment established that the 2.0 kb repeat is, in fact, the unit of amplification on the X. Comparison of the sequences of the twelve alpha X monomers allowed derivation of a 171 bp consensus sequence for alpha satellite DNA on the human X chromosome. These sequence data, combined with the results of filter hybridization experiments of total human DNA and X chromosome DNA, using subregions within the 2.0 kb repeat as probes, provide strong support for the hypothesis that individual human chromosomes are characterized by different alpha satellite families, defined both by restriction enzyme periodicity and by chromosome-specific primary sequence.     

蓝狐-卫星DNA的克隆及序列分析

利用限制性内切酶酶切蓝狐基因组, 经琼脂糖凝胶电泳, 对特异性亮带进行克隆、测序及序列分析。结果获得42个卫星DNA序列, 该卫星DNA单体大小为737 bp, G+C含量为51.9%, 单体之间同源性为91%~97%; 每个单体由3个约245 bp的亚重复串联构成, 亚重复之间的同源性为49%~55%; 在物种进化过程中, 该卫星DNA有G+C含量逐渐降低而A+T含量逐渐上升的趋势; 该卫星DNA为犬科动物种属所特有, 与犬着丝粒相关卫星DNA为同类卫星DNA, 同源性为74%, 命名为α-卫星DNA。