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PubMed1989

Detection of Trypanosoma cruzi by DNA amplification using the polymerase chain reaction.

The polymerase chain reaction was used to amplify a 188-base pair (bp) segment of the repetitive 195-bp nuclear DNA sequence of Trypanosoma cruzi that is the most abundant sequence in this organism. The reaction amplified this repetitive element in four T. cruzi isolates from widely separated geographic regions. No amplification of the 188-bp fragment occurred when DNAs extracted from Leishmania spp., African trypanosomes, or blood samples from mice and humans were used. Amplification of one-half of the DNA from a single T. cruzi parasite produced an amount of the 188-bp element that was readily visible in a gel stained with ethidium bromide. Hybridization of a radiolabeled probe to membrane-bound amplification products increased the sensitivity to a level at which 1/200 of the DNA in a single parasite could be detected. T. cruzi DNA was readily detected in DNA extracted from the abdominal contents of infected insect vectors reared in the laboratory. No parasite DNA was detected in the blood samples of two individuals known to be infected with T. cruzi, possibly because in such patients the number of circulating parasites are extremely low or because parasitemias are intermittent. These results represent a considerable increase in sensitivity over previously reported methods for the detection of T. cruzi infections. Polymerase chain reaction amplification can be used to evaluate large numbers of samples in a single day and thus should be useful in large-scale studies of the prevalence of T. cruzi in both insect vectors and mammalian hosts.

Animals↗
PubMed1997

PowerBLAST: a new network BLAST application for interactive or automated sequence analysis and annotation.

As the rate of DNA sequencing increases, analysis by sequence similarity search will need to become much more efficient in terms of sensitivity, specificity, automation potential, and consistency in annotation. PowerBLAST was developed, in part, to address these problems. PowerBLAST includes a number of options for masking repetitive elements and low complexity subsequences. It also has the capacity to restrict the search to any level of NCBI's taxonomy index, thus supporting "comparative genomics" applications. Postprocessing of the BLAST output using the SIM series of algorithms produces optimal, gapped alignments, and multiple alignments when a region of the query sequence matches multiple database sequences. PowerBLAST is capable of processing sequences of any length because it divides long query sequences into overlapping fragments and then merges the results after searching. The results may be viewed graphically, as a textual representation, or as an HTML page with links to GenBank and Entrez. For matching database sequences, annotated features are superimposed on the aligned query sequence in the output, thus greatly increasing the ease of interpretation. Such features may be used for automated annotation of new sequence because PowerBLAST output in ASN.1 form may be "dragged and dropped" into NCBI's Sequin program for sequence annotation and submission. PowerBLAST is capable of analyzing and annotating a 100-kb query in 60 min on NCBI's BLAST server.

Amino Acid Sequence↗
PubMed1991

Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

The gene responsible for cystic fibrosis, the most common severe autosomal recessive disorder, is located on the long arm of human chromosome 7, region q31-q32. The gene has recently been identified and shown to be approximately 250 kb in size. To understand the structure and to provide the basis for a systematic analysis of the disease-causing mutations in the gene, genomic DNA clones spanning different regions of the previously reported cDNA were isolated and used to determine the coding regions and sequences of intron/exon boundaries. A total of 22,708 bp of sequence, accounting for approximately 10% of the entire gene, was obtained. Alignment of the genomic DNA sequence with the cDNA sequence showed perfect colinearity between the two and a total of 27 exons, each flanked by consensus splice signals. A number of repetitive elements, including the Alu and Kpn families and simple repeats, such as (GT)17, (GATT)7, and (TA)14, were detected in close vicinity of some of the intron/exon boundaries. At least three of the simple repeats were found to be polymorphic in the population. Although an internal amino acid sequence homology could be detected between the two halves of the predicted polypeptide, especially in the regions of the two putative nucleotide-binding folds (NBF1 and NBF2), the lack of alignment of the nucleotide sequence as well as the different positions of the exon/intron boundaries does not seem to support the hypothesis of a recent gene duplication event. To facilitate detection of mutations by direct sequence analysis of genomic DNA, 28 sets of oligonucleotide primers were designed and tested for their ability to amplify individual exons and the immediately flanking sequences in the introns.

Amino Acid Sequence↗
PubMed1999

Analysis of the distribution of SIRE in the nuclear genome of Trypanosoma cruzi.

The short interspersed repetitive element (SIRE) of the nuclear genome of Trypanosoma cruzi was first detected when comparing the sequences of loci that encode the TcP2beta genes. The present study was designed to assess its distribution and organization in the nuclear genome of the parasite. Southern blots of genomic DNA from different strains demonstrated that each one possesses a defined and characteristic pattern of SIRE distribution. The conservation of the SIRE sequence in T. cruzi strains allowed the development of a rapid inter-SIRE PCR reaction that yields strain-specific amplicon profiles. In the T. cruzi CL Brener clone, we found 1500 copies of the element distributed in all chromosomes. 16 genomic fragments containing SIRE (SZs) were isolated and characterized. In fragments SZ10, SZ12 and SZ31, SIRE was linked to TcRel, a novel repeated sequence that constitutes the 3' end of vp85 genes. SIRE was also linked to an unknown open reading frame in fragments SZ14 and SZ23 which might be related to the subtelomeric regions of T. cruzi chromosomes. Further sequencing of SZ fragments revealed that SIRE was also linked to protein coding genes that have not yet been described in kinetoplastids such as the one coding for PRP22 helicase and a thimet oligopeptidase. To allow the rapid-generation genetic markers associated with SIRE, we developed a SIRE-bubble PCR reaction that provided several such markers for the construction of the physical map of chromosome XVI. The results herein demonstrate that SIRE-associated sites (SAS) may be of great help in physical mapping and interpretation of T. cruzi genomic sequence data.

Animals↗
PubMed2004

An intermediate grade of finished genomic sequence suitable for comparative analyses.

Although the cost of generating draft-quality genomic sequence continues to decline, refining that sequence by the process of "sequence finishing" remains expensive. Near-perfect finished sequence is an appropriate goal for the human genome and a small set of reference genomes; however, such a high-quality product cannot be cost-justified for large numbers of additional genomes, at least for the foreseeable future. Here we describe the generation and quality of an intermediate grade of finished genomic sequence (termed comparative-grade finished sequence), which is tailored for use in multispecies sequence comparisons. Our analyses indicate that this sequence is very high quality (with the residual gaps and errors mostly falling within repetitive elements) and reflects 99% of the total sequence. Importantly, comparative-grade sequence finishing requires approximately 40-fold less reagents and approximately 10-fold less personnel effort compared to the generation of near-perfect finished sequence, such as that produced for the human genome. Although applied here to finishing sequence derived from individual bacterial artificial chromosome (BAC) clones, one could envision establishing routines for refining sequences emanating from whole-genome shotgun sequencing projects to a similar quality level. Our experience to date demonstrates that comparative-grade sequence finishing represents a practical and affordable option for sequence refinement en route to comparative analyses.

Animals↗
PubMed1989

Characterization of a highly unstable mouse minisatellite locus: evidence for somatic mutation during early development.

A highly unstable mouse minisatellite locus, Ms6-hm, has been identified in mouse DNA fingerprints produced by cross-hybridization with human minisatellite probe 33.6. A 7-kb allele of Ms6-hm was cloned from a C57BL/6J mouse and collapsed to a 400-bp plasmid insert on propagation in Escherichia coli due to loss of the majority of minisatellite repeat units. Sequence analysis revealed that Ms6-hm has evolved by amplification within a member of the MT (mouse transcript) family of interspersed repetitive elements. Linkage analysis localized Ms6-hm near the brown coat color gene (b) on chromosome 4. Multiallelism and heterozygosity at this locus within inbred strains result from a high germline mutation rate to new-length alleles (2.5% per gamete). Mice mosaic for cells carrying a nonparental allele in somatic tissue, and in some cases also in the germline, provide evidence for additional, somatic, mutation events at Ms6-hm. In two mosaic mice the fraction of cells containing the nonparental allele has been shown to be indistinguishable in different adult tissues. These somatic mutation events at Ms6-hm must therefore occur very early in development, preceding the allocation of somatic lineages, and the same pool of primitive ectoderm cells must contribute equally to all somatic tissues. Under low-stringency hybridization conditions the collapsed subclone of Ms6-hm cross-hybridizes to other unstable loci in the mouse genome to generate a novel and highly individual specific mouse DNA fingerprint.

Alleles↗
PubMed1995

A novel minisatellite at a cloned hamster telomere.

The ends of eukaryotic chromosomes have special properties and roles in chromosome behavior. Selection for telomere function in yeast, using a Chinese hamster hybrid cell line as the source DNA, generated a stable yeast artificial chromosome clone containing 23 kb of DNA adjacent to (TTAGGG)n, the vertebrate telomeric repeat. The common repetitive element d(GT)n appeared to be responsible for most of the other stable clones. Circular derivatives of the TTAGGG-positive clone that could be propagated in E. coli were constructed. These derivatives identify a single pair of hamster telomeres by fluorescence in situ hybridization. The telomeric repeat tract consists of (TTAGGG)n repeats with minor variations, some of which can be cleaved with the restriction enzyme MnlI. Blot hybridization with genomic hamster DNA under stringent conditions confirms that the TTAGGG tracts are cleaved into small fragments due to the presence of this restriction enzyme site, in contrast to mouse telomeres. Additional blocks of (TTAGGG)n repeats are found approximately 4-5 kb internally on the clone. The terminal region of the clone is dominated by a novel A-T rich 78 bp tandemly repeating sequence; the repeat monomer can be subdivided into halves distinguished by more or less adherence to the consensus sequence. The sequence in genomic DNA has the same tandem organization in probably a single primary locus of >20-30 kb and is thus termed a minisatellite.

Animals↗
PubMed1984

Genomic structure and possible retroviral origin of the chicken CR1 repetitive DNA sequence family.

We have analyzed the sequence and structure of three CR1 family repetitive elements found in the region adjoining the 3' end of a chicken calmodulin gene. Members of this family are approximately equal to 300 base pairs long and are dispersed throughout the chicken genome. The present data, when taken together with that from four CR1s sequenced previously, reveal that the CR1 family has an overall structure possessing several features associated with the long terminal repeats of avian retroviruses. This finding implies that a retroviral mechanism may be responsible for the dispersion of CR1 sequences throughout the chicken genome. The seven different CR1 repeats that have been analyzed exist at defined locations in the chicken genome relative to nearby structural genes. A directional polarity has been assigned to the CR1 family based upon limited sequence homology to mammalian Alu-type sequences. Interestingly, whether present in 5' - or 3'-flanking DNA, the CR1 sequences have an inverse orientation such that they all "point toward" the nearby structural genes. This is consistent with the previously proposed concept that chicken CR1 sequences may be involved in defining the boundaries of active chromosomal domains of gene expression.

Animals↗
PubMed1995

Minisatellites corresponding to the human polycore probes 33.6 and 33.15 in the genome of the most 'primitive' known eukaryote Giardia lamblia.

DNA fingerprinting has been widely used for genetic characterization and individual recognition in a range of species, from man and other mammals down the evolutionary scale to some lower eukaryotic parasites. These techniques utilise repetitive elements first characterised in the human genome, known as minisatellites, which display extensive allelic variability. Few biological or biochemical characteristics have been found that distinguish isolates of Giardia lamblia (Gl), or their apparent variations in virulence. We have characterized 21 Gl isolates in axenic culture using DNA fingerprinting with the human minisatellite probes, 33.6 and 33.15. Up to 12 variable bands per isolate were recognized in the size range of 2.5 to 15 kb by Southern blot hybridization of restriction endonuclease-digested Gl DNA. Most isolates demonstrated a distinct banding pattern or DNA fingerprint. The results suggest that this method may provide a basis for the detailed genotypic characterization of Gl which will be amenable to computer and statistical analysis for use in studies of virulence and epidemiology. Also, as Gl occupies a unique phylogenetic position as a member of the earliest known divergence from the eukaryotic line of descent, this study may provide a useful model for the study of other important eukaryotic pathogens, as it is rapidly becoming apparent that minisatellites are ubiquitous components of eukaryotic genomes.

Animals↗
PubMed1999

Molecular structure and chromosomal localization of major repetitive DNA families in the chickpea (Cicer arietinum L.) genome.

Three major repetitive DNA sequences were isolated from a genomic library of chickpea (Cicer arietinum L.) and characterized with respect to their genomic organization and chromosomal localization. All repetitive elements are genus-specific and mostly located in the AT-rich pericentric heterochromatin. Two families are organized as satellite DNAs with repeat lengths of 162-168 bp (CaSat1) and 100 bp (CaSat2). CaSat1 is mainly located adjacent to the 18S rDNA clusters on chromosomes A and B, whereas CaSat2 is a major component of the pericentric heterochromatin on all chromosomes. The high abundance of these sequences in closely related species of the genus Cicer as well as their variation in structure and copy number among the annual species provide useful tools for taxonomic studies. The retrotransposon-like sequences of the third family (CaRep) display a more complex organization and are represented by two independent sets of clones (CaRep1 and CaRep2) with homology to different regions of Ty3-gypsy-like retrotransposons. They are distributed over the pericentric heterochromatin block on all chromosomes with extensions into euchromatic regions. Conserved structures within different crossability groups of related Cicer species suggest independent amplification or transposition events during the evolution of the annual species of the genus.

Amino Acid Sequence↗
PubMed1996

Details of retropositional genome dynamics that provide a rationale for a generic division: the distinct branching of all the pacific salmon and trout (Oncorhynchus) from the Atlantic salmon and trout (Salmo).

Salmonid species contain numerous short interspersed repetitive elements (SINEs), known collectively as the HpaI family, in their genomes. Amplification and successive integration of individual SINEs into the genomes have occurred during the evolution of salmonids. We reported previously a strategy for determining the phylogenetic relationships among the Pacific salmonids in which these SINEs were used as temporal landmarks of evolution. Here, we provide evidence for extensive genomic rearrangements that involved retropositions and deletions in a common ancestor of all the Pacific salmon and trout. Our results provide genetic support for the recent phylogenetic reassignment of steelhead and related species from the genus Salmo to the genus Oncorhynchus. Several other informative loci identified by insertions of HpaI SINEs have been isolated, and previously proposed branching orders of the Oncorhynchus species have been confirmed. The authenticity of our phylogenetic tree is supported both by the isolation of more than two informative loci per branching point and by the congruence of all our data, which suggest that the period between successive speciations was sufficiently long for each SINE that had been amplified in the original species to become fixed in all individuals of that species.

Animals↗
PubMed1986

Secondary genomic rearrangement events in pre-B cells: VHDJH replacement by a LINE-1 sequence and directed class switching.

We describe rearrangement events which alter expression from a productive VHDJH rearrangement in an Abelson murine leukemia virus-transformed pre-B cell line. One such rearrangement results in replacement of the initially expressed variable region gene by a site-specific join between the open reading frame of a LINE-1 repetitive element and a remaining JH segment. We discuss this event in the context of the 'accessibility' model of recombinase control, and with respect to similar rearrangements involved in oncogene activation. In another subclone of the same pre-B cell line, altered heavy chain expression resulted from a mu to gamma 2b class switch recombination which occurred by a recombination-deletion mechanism but involved a complex inversion. We provide evidence that the germline gamma 2b region is specifically expressed in pre-B cell lines and early in normal development. We propose that the predisposition of pre-B cell lines to switch to gamma 2b production may reflect a normal physiological phenomenon in which the switch event is directed by an increased 'accessibility' of the germline gamma 2b locus to switch-recombination enzymatic machinery. Our findings support the hypothesis that the apparently distinct recombination systems involved in variable region gene assembly and heavy chain class switching are both directed by the accessibility of their substrate gene segments.

Abelson murine leukemia virus↗
PubMed1994

An analysis of retroposition in plants based on a family of SINEs from Brassica napus.

The identification of a family of SINE retroposons dispersed in the genome of oilseed rape Brassica napus has provided the basis for an evolutionary analysis of retroposition in plants. The repetitive elements (called S1Bn) are 170 bp long and occupy roughly 500 loci by haploid genome. They present characteristic features of SINE retroposons such as a 3' terminal A-rich region, two conserved polymerase III motifs (box A and B), flanking direct repeats of variable sizes, and a primary and secondary sequence homology to several tRNA species. A consensus sequence was made from the alignment of 34 members of the family. The retroposon population was divided into five subfamilies based on several correlated sets of mutations from the consensus. These precise separations in subfamilies based on "diagnostic" mutations and the random distribution of mutations observed inside each subfamily are consistent with the master sequence model proposed for the dispersion of mammalian retroposons. An independent analysis of each subfamily provides strong evidence for the coexpression of at least three subfamily master sequences (SMS). In contrast to mammalian retroposition, diagnostic positions are not shared between SMS. We therefore propose that SMS were all derived from a general master sequence (GMS) and independently activated for retroposition after a variable period of random drift. Possible models for plant retroposition are discussed.

Base Sequence↗
PubMed1986

Molecular evolution of mammalian lactate dehydrogenase-A genes and pseudogenes: association of a mouse processed pseudogene with a B1 repetitive sequence.

A mouse genomic clone containing a lactate dehydrogenase-A (LDH-A) processed pseudogene and a B1 repetitive element was isolated, and a nucleotide sequence of approximately 3 kb was determined. The pseudogene and B1 element are flanked by perfect 13-bp repeats, and the B1 sequence starts at 14 nucleotides 3' to the presumptive polyadenylation signal of the pseudogene. The nucleotide sequences of the LDH-A genes and processed pseudogenes from mouse, rat, and human were compared, and a phylogenetic tree was constructed. The rate and pattern of nucleotide substitutions in the LDH-A pseudogenes are similar to previously reported results (Li et al. 1984). The average rate of nucleotide substitutions in the LDH-A pseudogenes is 4.3 X 10(-9)/site/year. The substitutions of C----T and G----A are most frequent, and A----G substitutions are relatively high. The rate of synonymous substitutions in the LDH-A genes is 5.3 X 10(-9), which is not significantly higher than the average rate of 4.7 X 10(-9) for 35 mammalian genes. The rate of nonsynonymous substitutions in the LDH-A genes is 0.20 X 10(-9), which is considerably lower than the average rate of 0.88 X 10(-9) for 35 mammalian genes. Thus, the mammalian LDH-A gene appears to be highly conserved in evolution.

Animals↗
PubMed1982

KpnI families of long, interspersed repetitive DNAs associated with the human beta-globin gene cluster.

KpnI families of long, interspersed repetitive DNAs are ubiquitous repetitive elements that occur in tens of thousands of copies in primate genomes. KpnI 1.2, 1.5 and two different KpnI 1.8-kb families were found within and flanking a 6.4-kb repeat beginning at 3 kb, 3' from the human beta-globin gene. Thus, six different types of KpnI families have now been identified, and four of these are found next to each other in a specific 6.4-kb repeat. Clones of the distinct KpnI families were hybridized to clones of the 6.4-kb repeat and adjacent sequences encompassed within some 17.6 kb of DNA lying 3' to the beta-globin gene cluster. The four KpnI families appear to make up the entire length of the 6.4-kb repeat. The linear order of the various cloned KpnI sequences in the repeat is 5'-pBK(1.8)26-pBK(1.5)54-pBK(1.2)11-pBK(1.8)11-3'. KpnI 1.2-kb sequences were also detected downstream from the 6.4-kb repeat. As in the case of the KpnI 1.2 and 1.5-kb families, the two KpnI 1.8-kb sequence families described here each hybridized with about 15% of all plaques in two independently generated human genome libraries.

DNA Restriction Enzymes↗
PubMed1997

Expressed var genes are found in Plasmodium falciparum subtelomeric regions.

The antigenic variation and cytoadherence of Plasmodium falciparum-infected erythrocytes are modulated by a family of variant surface proteins encoded by the var multigene family. The var genes occur on multiple chromosomes, often in clusters, and 50 to 150 genes are estimated to be present in the haploid parasite genome. Transcripts from var genes have been previously mapped to internal chromosome positions, but the generality of such assignments and the expression sites and mechanisms that control switches of var gene expression are still in early stages of investigation. Here we describe investigations of closely related var genes that occur in association with repetitive elements near the telomeres of P. falciparum chromosomes. DNA sequence analysis of one of these genes (FCR3-varT11-1) shows the characteristic two-exon structure encoding expected var features, including three variable Duffy binding-like (DBL) domains, a transmembrane sequence, and a carboxy-terminal segment thought to anchor the protein product in knobs at the surface of the parasitized erythrocyte. FCR3-varT11-1 cross-hybridizes with var genes located close to the telomeres of many other P. falciparum chromosomes, including a transcribed gene (FCR3-varT3-1) in chromosome 3 of the P. falciparum FCR3 line. The relatively high level transcription from this gene shows that the polymorphic chromosome ends of P. falciparum, which have been proposed to be transcriptionally silent, can be active expression sites for var genes. The pattern of the FCR3-varT11-1 and FCR3-varT3-1 genes are variable between different P. falciparum lines, presumably due to DNA rearrangements. Thus, recombination events in subtelomeric DNA may have a role in the expression of novel var forms.

Amino Acid Sequence↗
PubMedDate not supplied

Genotype analyses of Campylobacter isolated from the gastrointestinal tracts and the reproductive tracts of broiler breeder roosters.

Campylobacter is considered to be the leading bacterial etiologic agent of acute gastroenteritis in humans. Evidence implicates poultry as a major source of the organism for human illness; however, the pathways involved in Campylobacter contamination of poultry flocks, horizontal transmission and/or vertical transmission, remain unclear. Recent evidence implicates breeders as a potential source for Campylobacter contamination of the subsequent broiler offspring. In this investigation, Campylobacter isolated from feces, cloacal swabs, ceca, semen, and vas deferens of 12 breeder broiler roosters were genotyped by both flagellin A short variable region (flaA SVR) DNA sequence analysis and repetitive element (rep)-polymerase chain reaction (PCR). In 9 of 12 roosters, Campylobacter was isolated from multiple sites sampled. Comparison of multiple isolates obtained from individual roosters revealed variable results. In five of the nine roosters, all Campylobacter isolated demonstrated closely related flaA SVR DNA sequences as well as rep-PCR patterns; isolates from these roosters were collected from both the gastrointestinal and the reproductive tracts or from the gastrointestinal tract alone. The remaining four roosters had Campylobacter that were distinct by both typing methods. Isolates from two of these four roosters originated from both the gastrointestinal and the reproductive tracts. Isolates from the remaining two roosters originated from only the reproductive tract. Comparisons of all Campylobacter isolates recovered from a distinct sample type within either the reproductive tract or the gastrointestinal tract (feces, semen, cloaca, vas deferens, or ceca) were quite diverse. No relationship between the genotypes and the sample type could be ascertained. Further investigation is needed to determine the route of contamination and if the presence of Campylobacter within the rooster leads to contamination of the broiler offspring via the fertilized egg.

Animals↗
PubMed1985

The organization of two related subfamilies of a human tandemly repeated DNA is chromosome specific.

Several clones containing clusters of repetitive elements were isolated from a human chromosome 22 specific library. An EcoRI-XhoI fragment of 860bp was subcloned and was shown to belong to a family of tandemly repeated DNA linked to the Y-specific 3.4 kb HaeIII band. This probe hybridizes to several sets of sequences or subfamilies. The most abundant subfamily is a 1.8kb long sequence containing one EcoRV site, and in most repeats, one AvaII and one KpnI site. Using human-rodent somatic cell hybrid DNA, we have shown that this cluster is present on human chromosome 9 although presence on chromosome 15 is not excluded. Another subfamily, 6.1kb long, appears to be exclusive of chromosome 16. By in situ hybridization with metaphasic chromosomes, these sets of repeats were mapped to the constitutive heterochromatin of a few chromosomes. Coexistence in one genome of long tandem repeats of distinct organization but similar length may represent the outcome of a continuous process of fixation of variant sequences. Homologous repeats are also abundant in four higher primate genomes (Orangutan, gorilla, chimpanzee, and man) but absent in other primates (African green monkey, rhesus monkey, baboon, and mouse lemur).

Animals↗