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PubMed2002

Documenting ancient DNA quality via alpha satellite amplification and assessment of clone sequence diversity.

C/G-->T/A nucleotide alterations have been shown to hamper the straightforward interpretation of mitochondrial DNA sequence data derived from ancient tissues. Attempting to characterise this finding with respect to nuclear DNA, we contrasted two established protocols: (i) an enzymatic repair of damaged DNA, thereby translating and closing nicks in the DNA, and (ii) the application of N-phenacylthiazolium bromide, which cleaves glucose-derived protein crosslinks, presumably derived from Maillard reactions. We used medieval human bones that were refractory to standard PCR procedures. Due to negligible presence of short tandem repeat loci and also mitochondrial sequences, the extracted ancient DNA needed a higher copy PCR system to yield amplification products. The chosen PCR target was specific alphoid repetitive DNA with an experimentally determined minimum of 1000 copies per haploid genome. Alphoid repeat segments were generated from both contemporary DNA and DNA extracts of two human skeletons dating from 450-600 AD (omitting uracil N-glycosylase pre-treatment of the extracted samples), and were subsequently cloned and sequenced. The sequences were evaluated for the number and type of nucleotide alterations noted after the different pre-treatments, and were compared to our alphoid consensus sequence generated from modern DNA. Both methods failed to reflect the expected 32% variability among single alphoid repeats (accounting for locus-specific differences and polymerase errors) as well as to display the actual 2.88 ratio of transitions to transversions. Our data obtained from high-copy-number nuclear DNA mirror the phenomenon of sequence deviations observed in mitochondrial DNA extracted from old specimens.

Base Sequence↗

PubMed1993

Comparative diversity analysis of RFLPs and isozymes within and among populations of Hordeum vulgare ssp. spontaneum.

DNA restriction fragment length polymorphisms (RFLPs) and isozyme variation were surveyed in 268 accessions of a wild barley (Hordeum vulgare ssp. spontaneum) sampled from diverse ecogeographical areas in Israel and Iran. A total of 24 markers was used: 7 well characterized isozyme loci and 15 DNA probes which detected 17 putative loci and included three classes of DNA sequences (single copy, low copy and repetitive) representing all 7 barley chromosomes. Survey results indicated that both RFLPs and isozymes are highly polymorphic both within and among populations of this wild barley. The number of alleles per locus and average level of diversity do not differ between isozymes and RFLPs. However, the relative amounts of within vs. between population components vary greatly between these two sets of molecular markers. Isozymes demonstrated a larger amount of within population diversity, whereas RFLPs resolved a higher proportion of between population differentiation. Furthermore, RFLPs detected more heterozygosity than did isozymes. Both classes of markers resolved large numbers of multilocus combinations, the majority of which were represented by only one individual in the total sample. Up to 30% of the loci differ among individuals within populations, and about 50% of the loci differ among plants in different populations. While many important aspects of population diversity as determined by RFLPs are significantly correlated with those of isozymes, such correlation values are generally low, indicating that only a small proportion of the genetic variation detected by one class of markers can be predicted by the other.

Alleles↗

PubMed2000

Comparison of two amplification technologies for detection and quantitation of human immunodeficiency virus type 1 RNA in the female genital tract. Division of AIDS Treatment Research Initiative 009 Study Team.

Human immunodeficiency virus type 1 (HIV-1) RNA levels in female genital tract and peripheral blood samples were compared using two commercial amplification technologies: the Roche AMPLICOR HIV-1 MONITOR test and either the Organon Teknika nucleic acid sequence-based amplification (NASBA-QT) assay or the NucliSens assay. Estimates of HIV-1 RNA copy number were derived from internal kit standards and analyzed unadjusted and adjusted to a common set of external standards. We found a discordance rate of approximately 18% between the two technologies for the detection of HIV-1 in either the genital tract or peripheral blood samples. Detection discordance was not consistent among specimens or among women. There were no significant differences in adjusted or unadjusted estimates of HIV-1 RNA copy number in the genital tract samples using the AMPLICOR HIV-1 MONITOR test and either the NASBA-QT assay or the NucliSens assay. In addition, the estimated HIV-1 RNA copy number in peripheral blood samples did not differ when tested with the NucliSens assay and the AMPLICOR HIV-1 MONITOR test using kit standards. However, there was a significant difference in estimated RNA copy number between the NASBA-QT assay and the AMPLICOR HIV-1 MONITOR test for internal kit standards, which, as we have previously shown, was eliminated after adjustment with the external standards. Our results suggest that the Roche and Organon Teknika assays are equivalent for quantifying HIV-1 RNA in female genital tract specimens, although variation in detection does exist.

Cross-Sectional Studies↗

PubMed2006

The action of selection on codon bias in the human genome is related to frequency, complexity, and chronology of amino acids.

BACKGROUND: The question of whether synonymous codon choice is affected by cellular tRNA abundance has been positively answered in many organisms. In some recent works, concerning the human genome, this relation has been studied, but no conclusive answers have been found. In the human genome, the variation in base composition and the absence of cellular tRNA count data makes the study of the question more complicated. In this work we study the relation between codon choice and tRNA abundance in the human genome by correcting relative codon usage for background base composition and using a measure based on tRNA-gene copy numbers as a rough estimate of tRNA abundance. RESULTS: We term major codons to be those codons with a relatively large tRNA-gene copy number for their corresponding amino acid. We use two measures of expression: breadth of expression (the number of tissues in which a gene was expressed) and maximum expression level among tissues (the highest value of expression of a gene among tissues). We show that for half the amino acids in the study (8 of 16) the relative major codon usage rises with breadth of expression. We show that these amino acids are significantly more frequent, are smaller and simpler, and are more ancient than the rest of the amino acids. Similar, although weaker, results were obtained for maximum expression level. CONCLUSION: There is evidence that codon bias in the human genome is related to selection, although the selection forces acting on codon bias may not be straightforward and may be different for different amino acids. We suggest that, in the first group of amino acids, selection acts to enhance translation efficiency in highly expressed genes by preferring major codons, and acts to reduce translation rate in lowly expressed genes by preferring non-major ones. In the second group of amino acids other selection forces, such as reducing misincorporation rate of expensive amino acids, in terms of their size/complexity, may be in action. The fact that codon usage is more strongly related to breadth of expression than to maximum expression level supports the notion, presented in a recent study, that codon choice may be related to the tRNA abundance in the tissue in which a gene is expressed.

Amino Acids↗

PubMed2005

Genomewide comparative analysis of the highly abundant transposable element DINE-1 suggests a recent transpositional burst in Drosophila yakuba.

DINE-1 (Drosophila interspersed element) is the most abundant repetitive sequence in the Drosophila genome derived from transposable elements. It comprises >1% of the Drosophila melanogaster genome (DMG) and is believed to be a relic from an ancient transpositional burst that occurred approximately 5-10 MYA. We performed a genomewide comparison of the abundance, sequence variation, and chromosomal distribution of DINE-1 in D. melanogaster and D. yakuba. Unlike the highly diverged copies in the DMG (pairwise distance approximately 15%), DINE-1's in the Drosophila yakuba genome (DYG) have diverged by only 3.4%. Moreover, the chromosomal distribution of DINE-1 in the two species is very different, with a significant number of euchromatic insertions found only in D. yakuba. We propose that these different patterns are caused by a second transpositional burst of DINE-1's in the D. yakuba genome approximately 1.5 MYA. On the basis of the sequence of these recently transposed copies, we conclude that DINE-1 is likely to be a family of nonautomomous DNA transposons. Analysis of the chromosomal distribution of two age groups of DINE-1's in D. yakuba indicates that (1) there is a negative correlation between recombination rates and the density of DINE-1's and (2) younger copies are more evenly distributed in the chromosome arms, while older copies are mostly located near the centromere regions. Our results fit the predictions of a selection-transposition balance model. Our data on whole-genome comparison of a highly abundant TE among Drosophila sibling species demonstrate the unexpectedly dynamic nature of TE activity in different host genomes.

Animals↗

PubMed2000

[The construction of a standard RNA synthesized for quantitative RT-PCR system].

We constructed the standard RNA synthesized for the chimeric AML1-MTG8 transcripts and the house-keeping gene, glyceraldehyde-3-phosphate dehydrogenase(GAPDH) transcripts in real-time quantitative RT-PCR system. AML-MTG8 transcripts was detectable in 10 fg of synthetic RNA(3.5 x 10(3) copies). Linearity was from 3.5 x 10(3) to 3.5 x 10(9) copies. Threshold cycle(CT) is defined as the fractional cycle number at which the reporter fluorescence generated by cleavage of the probe passes a fixed threshold above baseline. The standard curve, where the known amounts of RNAs were used, showed a good correlation between the copies of AML1-MTG8 RNA and CT(r = -0.995). The within-run and day-to-day coefficients of variation(CV) in AML1-MTG8 RNA by this system were 9.5-24.7%(n = 10) and 21.7-42.2% (n = 8), respectively. GAPDH transcripts was detectable in 10 fg of synthetic RNA(6.1 x 10(4) copies). Linearity was from 6.1 x 10(4) to 6.1 x 10(8) copies. The standard curve, where the known amounts of RNAs were used, showed a good correlation between the copies of GAPDH RNA and CT(r = -0.993). The within-run and day-to-day CV in GAPDH RNA by this system were 9.3-14.6%(n = 10) and 14.7-15.8% (n = 10), respectively. Thus, we suggested that synthesized RNA as a standard RNA may be useful in quantitative RT-PCR for clinical application.

Core Binding Factor Alpha 2 Subunit↗

PubMed1989

Frequent alteration of the DF3 tumor-associated antigen gene in primary human breast carcinomas.

The gene for the human DF3 breast carcinoma-associated antigen contains a conserved (G + C)-rich 60-base pair tandem repeat and maps to chromosome 1q21-24. In the present study we isolated and characterized 1220 base pairs of nonrepetitive adjacent sequences. Multiple alleles were identified by fragment size. Signal intensity of hybrids with the tandem and unique sequence probes indicated that allelic variation is due to different numbers of repeats. Probes for both the tandem and the unique sequences were used to study the DF3 locus in human breast tumor DNAs. Seventy of 110 breast tumor DNAs were informative at the DF3 locus. Of these, 20 (29%) showed a loss of heterozygosity, while eight (11%) had an increased copy number of one allele. In some cases, the loss of heterozygosity or increased copy number did not extend to other markers on chromosome 1q or 1p. These data indicate that the chromosomal region around the DF3 locus is affected by mutations at high frequency.

Amino Acid Sequence↗

PubMed1983

Bovine papillomavirus type 1 genome in hamster sarcoma cells in vivo and in vitro: variation in the level of transcription.

The physical state and expression of the bovine papillomavirus type 1 (BPV-1) genome were analysed in two transplantable hamster sarcomas (HT1 and HT2) after a low number of transplantations, in two tumourigenic cell lines derived from the first transplant of HT2 sarcoma and another transplantable sarcoma (HT3) and in tumours obtained by grafting these cells. Blot hybridization experiments indicated the presence of multiple free copies of the whole viral genome in HT1 and HT2 tumour transplants (100 and 25 copies/cell respectively), irrespective of the number of in vivo or in vitro passages. In contrast, HT3 cells, and tumours induced by these cells, at early and late passages in vitro contained viral sequences probably integrated in the cell genome, in addition to the free viral DNA sequences. Polyadenylated viral transcripts were easily detected in HT1, HT2 and HT3 tumours obtained at early passages in vivo and in vitro, with electrophoretic mobilities corresponding to 1200 to 1400 bases (HT1, HT2 and HT3), 1750 bases (HT1) and 2500 bases (HT2). Homologous sequences of the transcripts were localized in the transforming BamHI-HindIII fragment of BPV-1 DNA, mainly in the BamHI-EcoRI fragment (0.31 to 0.602 map units). In contrast, almost no viral transcription was detected in HT2 and HT3 cells after 50 subcultures and in the tumours induced by these cells. This suggests that the tumourigenicity of the HT2 and HT3 cells is compatible with a very low level of expression of the transforming region of the BPV-1 genome.

Animals↗

PubMed1972

Virus deoxyribonucleic acid sequences in subdiploid and subtetraploid revertants of polyoma-transformed cells.

Polyoma-transformed cells can revert in the properties characteristic of transformation, although they maintain the polyoma-specific T antigen. Transformed cells contain the same number of copies of polyoma virus deoxyribonucleic acid (DNA) per cell (eight) as revertants with a subdiploid or a subtetraploid chromosome number. The results indicate that the duplication of chromosomes in the subtetraploid revertants did not include the chromosomes that carry the viral genome. The virus DNA in both transformed and revertant cells was associated with high-molecular-weight cell DNA. Reversion of the properties of transformed cells was, therefore, not associated either with a decrease in number of virus DNA copies per cell or with a lack of association of the virus DNA with cell DNA.

Animals↗

PubMed1994

Strain variation in the katG region of Mycobacterium tuberculosis.

Southern blot analysis of chromosomal DNA from clinical isolates of Mycobacterium tuberculosis using cosmid DNA probes revealed extensive strain variation in the katG region of the genome. In addition to deletion of the katG gene itself in some isoniazid-resistant strains, adjacent DNA fragments were missing or altered in a range of drug-sensitive and drug-resistant isolates. A species-specific 2kb Kpnl fragment located 10kb upstream of katG in M. tuberculosis H37Rv hybridized to fragments of differing size in different clinical isolates and was characterized in detail. Sequence analysis of this fragment in detail. Sequence analysis of this fragment showed that it comprised three tandem copies of a novel 75 bp repeat element flanked by multiple copies of the previously described 10 bp major polymorphic tandem repeat of M. tuberculosis (MPTR). The copy number of the 75 bp repeat was found to vary between strains, allowing application of a polymerase chain reaction amplification strategy for strain differentiation. These results indicate that the katG region of the M. tuberculosis genome is highly variable and unstable. The presence of repetitive sequences may contribute to instability in this region of the genome.

Amino Acid Sequence↗

PubMed1995

Mycoplasma hyorhinis vlp gene transcription: critical role in phase variation and expression of surface lipoproteins.

Mycoplasma hyorhinis contains clustered vlp genes encoding variable lipoproteins (Vlps), the major coat proteins and surface antigens of this wall-less prokaryotic pathogen. vlp genes are subject to discrete, high frequency mutations independently affecting the size or the expression of variant Vlp products. Change in Vlp size occurs by mutations altering the number of tandem intragenic repeats at the 3' end of each single-copy vlp gene. In this report, phase-variant Vlp expression is shown to result from altered vlp gene transcription. vlpA, vlpB and vlpC transcripts were monitored in a clonal lineage selected to display various Vlp phenotypes. Each vlp gene was expressed as a distinct transcript, which was subject to drastic ON/OFF switches associated with random insertion/ deletion mutations in a homopolymeric tract of adenine residues in the promoter region of all vlp genes. Unexpectedly, the level of vlp transcripts appeared to depend on the length of the corresponding genes in the ON configuration. Higher proportional levels of shorter vlp transcripts were shown to reflect a greater abundance of short Vlp lipoproteins present in L-[35S]-cysteine-labelled membrane protein preparations. The vlp cluster provides a heritable, and highly mutable, locus for the generation of surface diversity through random promoter mutations affecting the expression of genes, whose products also vary in length and abundance, by virtue of separate mutations in structural regions of the genes.

Antigens, Bacterial↗

PubMed1992

An investigation of the effect of antisense RNA gene on bovine leukaemia virus reproduction in cell culture.

A possible approach to control of bovine lymphoproliferative disease caused by bovine leukaemia virus (BLV) may be the development of an "antiviral information immunity" based on the effect of anti-sense RNA (asRNA). A numbers of constructs were obtained, under control of various promotors (herpesvirus thymidine kinase, T-antigen SV40 promoter), carrying as DNA against gene X, the expression product of which is a transactivator of viral transcription from the BLV LTR promotor. As a model system for the analysis of antiviral activity of constructs developed, cloned continuous cell lines of BLV-producing FLK cells were used. The level of BLV expression in cells transfected with the constructs was determined by various parameters. Differences were detected in different clones obtained from non-transfected cells, as well as variation between transfected clones, as measured by reverse transcriptase, competitive radio-immunoassay for BLV p24, the viral particle count on agar membrane, and the tumorigenicity for nude mice. The differences in inhibition of expression of BLV genes and their products may be explained in terms of the site of integration of asDNA and the number of integrated copies.

Animals↗

PubMed2005

Development of a sensitive and quantitative diagnostic assay for fish nervous necrosis virus based on two-target real-time PCR.

The aim of the present work was to develop two new independent SYBR Green I-based real-time PCR assays for both detection and quantification of betanodavirus, an RNA virus that infects several species of marine teleost fish causing massive mortalities in larvae and juveniles. The assays utilized two pairs of primers targeting highly conserved regions of both the RNA molecules forming the betanodavirus genome: RNA1 encoding the RNA-dependent RNA polymerase (RdRP) and RNA2 encoding the coat protein (CP). The specificity of amplifications was monitored by the melting analysis and agarose gel electrophoresis of the amplified products. The applicability of these assays was confirmed with 21 betanodavirus strains, covering all the four main clades. In addition, a BLAST (NCBI) search with the primer sequences showed no genomic cross-reactivity with other viruses. The new assays were able to quantify concentrations of betanodavirus genes ranging from 10(1) to 10(8) copies per reaction. The intra-assay coefficients of variation (CV) of threshold cycle (Ct) values of the assays were 1.5% and 1.4% for CP and RdRP RNAs, respectively. The inter-assay CVs of Ct values were 2.3% and 2.4% for CP and RdRP RNAs, respectively. Moreover, regression analysis showed a significant correlation (R2>0.97) between genome number, as determined by real-time PCR assays and the corresponding virus titer expressed as TCID50/ml of two different betanodavirus strains propagated in cell culture. The two assays were compared with a previously established one-step RT-PCR assay and with the classical virus isolation test and found to be more sensitive. In conclusion, the developed real-time RT-PCR assays are a reliable, specific and sensitive tool for the quantitative diagnosis of betanodavirus.

Animals↗

PubMed1995

Listeria monocytogenes exists in at least three evolutionary lines: evidence from flagellin, invasive associated protein and listeriolysin O genes.

Regions of the genes encoding flagellin (flaA), the invasive associated protein (iap), listeriolysin O (hly) and 23S rRNA were sequenced for a range of Listeria monocytogenes isolates of different origin and serotypes. Several nucleotide sequence variations were found in the flaA, iap and hly genes. No differences were found for the rRNA genes, but our approach does not exclude the existence of differences between single copies of these genes. Based on the sequence differences, the L. monocytogenes strains can be divided into three distinct sequence types. Further, the presence of only a small number of sequence differences within each group indicates a strong degree of conservation within the groups. There was a complete correspondence among the groups of strains formed according to the analysis of the flaA, iap and hly genes, and the grouping correlates with serotype, pulsed field gel electrophoretic and multilocus enzyme electrophoretic data. Analysis of the region encoding the threonine-asparagine repeat units in the iap gene revealed some striking features. Sequence type 1 strains were found to have 16-17 repeats, sequence type 2 strains had 16-20 repeats whereas the two sequence type 3 strains analysed had only 11 repeats. Furthermore, within a 19 bp segment there was a 37% difference between the sequences of type 1 and 2 strains and that segment was absent in type 3 strains. Within the threonine-asparagine repeat region the nucleotide differences gave rise to four amino acid changes; however, all were changes among the three amino acids present in the repeat structure indicating a strong selective pressure on the composition of this region.

Animals↗

PubMed2000

Association analysis of 5HT transporter gene in bipolar disorder in the Indian population.

A variable number tandem repeat (VNTR) polymorphism consisting of multiple copies of a 17-bp repeat in the second intron of the serotonin transporter gene (SERT) has been reported. Different alleles of this VNTR have been found to be associated with bipolar disorder and schizophrenia. These findings have been confirmed in some populations, but disconfirmed in others. Furthermore, significant ethnic variations in the distribution of these alleles both in normal and patient populations also have been reported. We analyzed the VNTR polymorphism in 50 Indian patients with bipolar disorder and in ethnically matched controls. Two alleles corresponding to 10 and 12 repeats of the VNTR were found in both groups. There were no significant differences either in allele frequency or genotype frequency between the two groups. The nine-repeat allele that has been reported in Japanese and Caucasian populations was absent in our sample. Although it will be important to extend the present study in a larger sample, our initial results do not suggest any large association with alleles of the VNTR in the SERT gene and bipolar disorder in Indian patients. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:170-172, 2000.

Alleles↗

PubMed1991

Minisatellite-like repeat sequences in the genome of rodent malaria parasites.

Using minisatellite DNA probes and various Southern blots containing DNA samples from rodent malaria parasites it was shown that minisatellite-like sequences occur in the genome of these Plasmodium species. In contrast to the high copy number as observed in higher eukaryotes, the use of fingerprinting techniques on DNA from these parasites reveals that minisatellite sequences are only present at a small number of loci. When parasite lines which differ in biological parameters are compared, no frequent restriction fragment length polymorphism (RFLP) is observed. Screening data banks revealed the presence of repeated copies of one of the probes, which has the monomer sequence CAGGTGG, in the DNA encoding the immunodominant peptide repeat region of the circumsporozoite (CS) protein from a Plasmodium cynomolgi strain. Comparing the sequences for the CS protein from a number of strains of P. cynomolgi revealed that the core region of the repeats, though subject to limited variation, shares homology to the bacterial recombination signal sequence Chi (GCTGGTGG). The implications of the above findings for genetic variation in malaria parasites and evolution of minisatellite sequences will be discussed.

Animals↗

PubMed2004

Genotypic variation and stability of four variable-number tandem repeats and their suitability for discriminating strains of Mycobacterium leprae.

It has not been possible to distinguish different strains of Mycobacterium leprae according to their genetic sequence. However, the genome contains several variable-number tandem repeats (VNTR), which have been used effectively in strain typing of other bacteria. To determine their suitability for differentiating M. leprae, we developed PCR systems to amplify 5 different VNTR loci and examined a battery of 12 M. leprae strains derived from patients in different regions of the United States, Brazil, Mexico, and the Philippines, as well as from wild armadillos and a sooty mangabey monkey. We found diversity at four VNTR (D = 0.74), but one system (C(16)G(8)) failed to yield reproducible results. Alleles for the GAA VNTR varied in length from 10 to 16 copies, those for AT(17) varied in length from 10 to 15 copies, those for GTA varied in length from 9 to 12 copies, and those for TA(18) varied in length from 13 to 20 copies. Relatively little variation was seen with interspecies transfer of bacilli or during short-term passage of strains in nude mice or armadillos. The TA(18) locus was more polymorphic than other VNTR, and genotypic variation was more common after long-term expansion in armadillos. Most strain genotypes remained fairly stable in passage, but strain Thai-53 showed remarkable variability. Statistical cluster analysis segregated strains and passage samples appropriately but did not reveal any particular genotype associable with different regions or hosts of origin. VNTR polymorphisms can be used effectively to discriminate M. leprae strains. Inclusion of additional loci and other elements will likely lead to a robust typing system that can be used in community-based epidemiological studies and select clinical applications.

Genetic Variation↗

PubMed1981

Human inherited marker chromosome 22 short-arm enlargement: investigation of rDNA gene multiplicity, Ag-band size, and acrocentric association.

The banding characteristics of an extreme variant familial chromosome 22 short-arm enlargement are described. Ag-AS staining for nucleolar-organizer regions, identified two areas of rDNA actively coding for 18S and 28S rRNA, the one being a broad distal Ag-band and the other a narrower centromeric Ag-band. The DNA in the major portion of the enlarged short arm was highly methylated, as shown by the binding of antibodies to 5-methylcytidine after UV-denaturation of chromosomal DNA. Mean Ag-band size on the aberrant 22p+ correlated with the mean number of 22p+ associations. Association of 22p+ was no greater than that of other acrocentrics, in spite of a presumed excess number of rDNA gene copies. This case represents only the second such normal variant defined by these techniques.

Cell Division↗