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PubMed1992

Chromosome-size variation in Giardia lamblia: the role of rDNA repeats.

Giardia lamblia trophozoites contain at least five sets of chromosomes that have been categorized by chromosome-specific probes. Pulsed field separations of G. lamblia chromosomes also demonstrated minor bands in some isolates which stained less intensely with ethidium than the major chromosomal bands. Two of the minor bands of the E11 clone of the ISR isolate, MBa and MBb, were similar to each other and to chromosomal band I by hybridization to total chromosomal DNA and by hybridization of specific probes. In order to determine the extent of this similarity, I have developed a panel of probes for many of the Pacl restriction fragments and have shown that most of the Pacl and Notl fragments found in MBa are also present in MBb. The differences are found in both telomeric regions. At one end, MBb contains a 300 kb region not found in MBa. At the other end of MBb is a 160 kb region containing the rDNA repeats which is bounded on one end by the telomeric repeat and on the other by sites for multiple enzymes that do not digest the rDNA repeats. The corresponding region of MBa is 23 kb in size. The size difference is consistent with the eightfold greater number of rDNA repeats in MBb than MBa and suggests that 30% of the size difference is accounted for by different numbers of copies of the rDNA repeat. MBa of another ISR clone (ISR G5) is 150 kb larger in size than MBa of ISR E11. The data suggest that MBa and MBb are homologous chromosomes of different sizes and that a portion of the size difference is accounted for by different copy numbers of the rDNA repeat.

Animals↗

PubMed1994

Binding of nuclear factors to a satellite DNA of retroviral origin with marked differences in copy number among species of the rodent Ctenomys.

The major satellite DNA of the subterranean rodent Ctenomys, named RPCS, contains several consensus sequences characteristic of the U3 region of retroviral long terminal repeats (LTRs), such as a polypurine tract, CCAAT boxes, binding sites for the CCAAT/enhancer-binding protein (C/EBP), a TATA box and putative polyadenylation signals. RPCS presents an enormous variation in abundance between species of the same genus: while C. australis or C. talarum have approximately 3 x 10(6) copies per genome, C. opimus has none. A sequence (RPCS-I) with identity to the SV40-enhancer core element, present in all the repeating units of the satellite is specifically protected in DNase I footprintings. Competitions of band-shift assays with different transcription factor binding sites indicate that binding to RPCS-I is specific and involves CCAAT proteins related to NF-1, but not to C/EBP. By the use of quantitative protein/DNA binding assays we determined that, despite of their conspicuous difference in RPCS copy number, C. talarum and C. opimus have equivalent amounts and identical quality of RPCS-binding proteins. These results are consistent with the observation, by in situ hybridization, that RPCS is clustered in heterochromatic regions, where it might have restricted accessibility to transcription factors in vivo. This is the first report of the binding of transcription factors to a satellite DNA of retroviral origin.

Animals↗

PubMed2003

Fingerprints as evidence for a genetic profile: morphological study on fingerprints and analysis of exogenous and individual factors affecting DNA typing.

Material recovered from 374 fingerprints left by eleven laboratory workers on three different substrates (glass, wood, metal) at a standard pressure time of 30 s, with and without preliminary handwashing, was submitted to morphological, quantitative, and type analysis. Morphological and agarose-gel electrophoresis analysis showed that a non-negligible amount of epidermal corneal cells presented apoptotic alterations. The quantity of DNA recovered from fingerprints ranged between 0.04 to 0.2 ng, and in a significant number of experiments no DNA was detected. Handwashing reduced the amount of DNA recovered from fingerprints. The "shedder status" of the donor was a very important factor, causing inter-individual variations in the amount of DNA left by fingerprints. Spurious alleles from laboratory-based and secondary transfer contamination, stutters, and other artifacts described when analyzing low-copy-number DNA and capable of affecting correct profiles were observed.

Alleles↗

PubMed2000

Molecular dissection of Mycoplasma hominis.

M. hominis is commonly found as part of the normal flora in the female genital tract, but several studies have shown that it may be involved in a variety of urogenital infections. The basis for clinical manifestations in some patients has varyingly been attributed to host and M. hominis factors. The host factors involved in the infection process are largely unknown. M. hominis have no cell wall and outer membranes, and at present it seems plausible that M. hominis possesses genetic systems allowing the bacteria in vivo to alter its antigenic structure on the membrane surface and consequently circumvent the host immune system. The studies of M. hominis have shown that the antigenic variation is pronounced between surface exposed membrane proteins from different isolates. The genetic background for this variation has been investigated for three surface exposed membrane proteins: P120, Lmp, and Vaa. P120 and P120' are similar proteins in M. hominis without any homology to other known proteins. A hypervariable region in the otherwise conserved P120 protein seems to be very antigenic in patients with immunologically verified M. hominis infection. The remaining part of P120 as well as the entire P120' protein do not seem to elicit significant antibody formation. Two genes in M. hominis, lmp1 and lmp3, contain numerous highly similar 0.5 kb tandem repeats at their 3'-end. The proteins, Lmp1 and Lmp3, are synthesized from the lmp1 and lmp3 genes, respectively. Lmp1 shows size variation among M. hominis isolates. M. hominis isolates investigated in detail show that the size variation of Lmp1 corresponds to the variation in number of 0.5 kb repeats contained within the lmp1 gene. Lmp3 appears to have a lesser tendency to size variation. M. hominis isolates were found with deletions involving the lmp1 stop codon leading to translation of the downstream gene lmp2 and expression of a chimeric Lmp1-Lmp2 protein. The number of repeated elements in the lmp1 gene of a M. hominis isolate correlates with the extent of anti-Lmp antibody induced agglutination between the bacteria. Vaa is a protein involved in cell adherence. vaa is a single copy gene containing tandem repeated elements like the lmp gene family. The number of repeats in the Vaa protein differs between M. hominis isolates leading to size variation. It has been suggested that the number of repeated elements is of importance in the bacteria-host adhesion process. Beside the size variation Vaa demonstrates phase variation due to frequent frame shift mutation in a specific region near the 5'-end of the structural gene. Based on the investigations of M. hominis and other mycoplasmas several genetic mechanisms seem to be responsible for the antigenic variation of surface exposed membrane proteins in mycoplasmas: 1) variation in protein size due to insertions or deletion of repeated elements in the structural gene, 2) presence of multi-gene families, and 3) phase variation due to mutations in the promotor region or the coding region. The influence of specific antibodies on antigenic variation of membrane proteins has not been studied in greater detail in mycoplasmas. In M. hominis it was investigated whether the presence in the culture medium of monoclonal antibodies directed against the repeated elements in the M. hominis Lmp proteins would affect gene structure and consequently protein expression. The presence of anti-Lmp antibodies resulted in overgrowth of bacteria with specific deletions in the repeated elements of lmp1 leaving the lmp3 gene unchanged. The precise mechanism leading to the dominance of M. hominis isolates with fewer 0. (ABSTRACT TRUNCATED)

Amino Acid Sequence↗

PubMed1985

Sister chromatid exchange and the evolution of rDNA spacer length.

The structures of rDNA spacers from several species have been characterized and virtually all have internally repeated sequences. Different numbers of these internal repeats are responsible for most spacer length variation. Because unequal recombination between these internal repeats will cause new length variation, while unequal exchange between rDNA copies will homogenize the variants, we modeled the interaction of these two processes. Two models were used to simulate both types of unequal exchange at the sister chromatid level. Both models indicate that a narrow range of relative recombination frequencies is required to produce levels of variability comparable to those published. One model puts a lower limit on the number of internal repeats, and the other puts both a lower and upper limit on the number of repeats. The model with both maximum and minimum constraints produces a distribution closer to actual spacer distributions. These results imply that small changes in recombination rates can generate the differences in numbers of length variants observed in different species.

Animals↗

PubMed1994

Quantification of mRNA in human tissue using fluorescent nested reverse-transcriptase polymerase chain reaction.

We report the development of a quantitative nested reverse-transcriptase polymerase chain reaction which utilizes a fluorescence detection system. Using specific primer pairs to study mRNA for endothelin receptors in the human kidney, we synthesized a cRNA construct containing the same sequences but yielding a PCR product some 300 base pairs larger than native mRNA. Inclusion of a known amount of construct as internal standard with tissue RNA prior to cDNA synthesis allowed all reactions to occur under the same conditions in the same tube. In the nested PCR reaction, serial dilutions made before the second round enabled construction of a standard curve for each assay, and confirmation that standard and sample curves remained parallel. This indicates that both cDNAs amplified at the same rate. One internal primer was fluorescently labeled. Quantification of products using an ABI 373A sequencer with Genescan software gave sensitive and reproducible results. Analysis of a needle biopsy (10 mg) of histologically normal cortex gave 0.4 amol ETA mRNA and 1.6 amol ETB mRNA/micrograms total RNA. In medulla these values were 0.46 and 1.16 amol/micrograms, respectively. Ratios of ETB to ETA message were 74:26 in cortex and 77:23 in medulla, agreeing with previous ligand binding studies of receptor protein. Intra- and interassay coefficients of variation were 4.5 and 5.3%. This new method has potential for widespread application to the study of low copy-number mRNA or where only very small amounts of tissue are available, such as biopsy specimens.

Base Sequence↗

PubMed2000

Quantification of total genomic DNA and selected repetitive sequences reveals concurrent changes in different DNA families in indica and japonica rice.

This paper describes a fluorescence in situ hybridization (FISH) analysis of three different repetitive sequence families, which were mapped to mitotic metaphase chromosomes and extended DNA fibers (EDFs) of the two subspecies of rice (OrYza sativa), indica and japonica (2n = 2x = 24). The repeat families studied were (1) the tandem repeat sequence A (TrsA), a functionally non-significant repeat; (2) the [TTTA-GGG]n telomere sequence, a non-transcribed, tandemly repeated but functionally significant repeat; and (3) the 5S ribosomal RNA (5S rDNA). FISH of the TrsA repeat to metaphase chromosomes of indica and japonica cultivars revealed clear signals at the distal ends of twelve and four chromosomes, respectively. As shown in a previous report, the 17S ribosomal RNA genes (17S rDNA) are located at the nucleolus organizers (NORs) on chromosomes 9 and 10 of the indica cultivar. However, the japonica rice lacked the rDNA signals on chromosome 10. The size of the 5S rDNA repeat block, which was mapped on the chromosome 11 of both cultivars, was 1.22 times larger in the indica than in the japonica genome. The telomeric repeat arrays at the distal ends of all chromosome arms were on average three times longer in the indica genome than in the japonica genome. Flow cytometric measurements revealed that the nuclear DNA content of indica rice is 9.7% higher than that of japonica rice. Our data suggest that different repetitive sequence families contribute significantly to the variation in genome size between indica and japonica rice, though to different extents. The increase or decrease in the copy number of several repetitive sequences examined here may indicate the existence of a directed change in genome size in rice. Possible reasons for this phenomenon of concurrent evolution of various repeat families are discussed.

Blotting, Southern↗

PubMed1990

Construction and hormone regulation of a novel retroviral vector.

We report the analysis of a self-inactivating retroviral vector, constructed to allow inducible gene expression of inserted sequences from the mouse mammary tumour virus hormonal response element. The cloning strategy has been designed to allow for ease of insertion of the genes of interest. The vector contains the aph gene, allowing geneticin-resistance selection in mammalian cells. We have characterised dexamethasone (Dex)-induced increase in gene expression using the reporter gene encoding chloramphenicol acetyltransferase (CAT) inserted into the retroviral vector. We observe low basal levels of CAT activity in infected cells which is increased up to 50-fold by induction with Dex. The induction of pooled clones is 13.3-fold. Variation in Dex-induced CAT activity is observed in independently infected clones, which is not explained by proviral copy number.

Animals↗

PubMed2001

Detection and growth kinetics of SV40 preparations with different enhancer element copy number.

It has been reported that the rate of growth of SV40 in certain cell culture systems is affected by the number of copies of the enhancer element, and that this might in turn affect the ability of manufacturers of polio vaccines to detect SV40 contamination in tests for adventitious agents. Eleven strains of SV40 of which three were primary isolates, three were control strains used in different laboratories and five were well characterised were examined for their enhancer copy number. It was found that the three primary isolates contained a single copy while three preparations used as controls in different laboratories were mixtures of single and multiple copy strains. The remaining five strains consisted of one known double enhancer and four known single enhancer strains. All eleven strains were titrated in BSC-1 cells. There was no correlation between the number of copies of the enhancer element and the rate of growth or the final titre reached, although there was variation between the strains.

Animals↗

PubMed2003

Longitudinal monitoring of 2-long terminal repeat circles in peripheral blood mononuclear cells from patients with chronic HIV-1 infection.

OBJECTIVE: To evaluate the potential use of 2-long terminal repeats (LTR) HIV circular DNA quantification for the monitoring of ongoing virus replication in treated HIV-1-infected patients. DESIGN AND METHODS: In a longitudinal setting, where the natural course of HIV-1 infection was in most cases disrupted by continuous or discontinuous antiviral therapy, 2-LTR circles of HIV-1 DNA were quantified in serial peripheral blood mononuclear cell samples, selected in retrospect from 16 patients with chronic HIV-1 infection, using quantitative real-time PCR. We compared variations of 2-LTR circle level with concomitant variations in plasma viral RNA level and with the frequency of productively infected cells and chromosome associated proviral DNA copy numbers in patient's peripheral blood mononuclear cells. RESULTS: Antiviral treatment led to a sharp decrease in plasma viraemia and infectious cell frequency. In contrast, we found that levels of proviral DNA and 2-LTR circles were significantly lower under treatment only when groups of specimens that were homogeneous, with respect both to plasma viraemia and infectious cell frequency, were compared. Moreover, during the time of undetectable plasma viraemia, scarcely any decline in proviral DNA or 2-LTR circle levels was observed. CONCLUSIONS: The low impact of antiviral treatment on 2-LTR circle levels in vivo, when plasma viraemia and infectious cell frequency both dramatically decline lead us to conclude that 2-LTR circles should not be used for the monitoring of recent viral replication in treated patients.

Anti-HIV Agents↗

PubMed2004

Conserved genetic basis of a quantitative plumage trait involved in mate choice.

A key question in evolutionary genetics is whether shared genetic mechanisms underlie the independent evolution of similar phenotypes across phylogenetically divergent lineages. Here we show that in two classic examples of melanic plumage polymorphisms in birds, lesser snow geese (Anser c. caerulescens) and arctic skuas (Stercorarius parasiticus), melanism is perfectly associated with variation in the melanocortin-1 receptor (MC1R) gene. In both species, the degree of melanism correlates with the number of copies of variant MC1R alleles. Phylogenetic reconstructions of variant MC1R alleles in geese and skuas show that melanism is a derived trait that evolved in the Pleistocene.

Animals↗

PubMed1989

Interphase cytogenetics using biotin and digoxigenin labelled probes I: relative sensitivity of both reporter molecules for detection of HPV16 in CaSki cells.

This study was undertaken to develop technology for the detection of nucleic acid using two different DNA probe reporter molecules, the ultimate aim being to differentially label two nucleic acids within the same nucleus. Digoxigenin and biotin were used to label DNA probes. The absolute and relative sensitivity of digoxigenin and biotin labelled DNA probes for detecting integrated human papilloma virus 16 (HPV16) was investigated in CaSki cells by non-isotopic in situ hybridisation (NISH). Several methods for the detection of labelled probes were also investigated. The optimal sensitivity of digoxigenin labelled probe was equivalent to that of biotin when alkaline phosphatase was used as the final detector. The median number of discrete viral signals discernible in each cell with the most sensitive detection system was seven to eight with both labelled probes. The average number of HPV16 genomes in each CaSki cell, derived by dot blot hybridisation, was about 270. The calculated absolute sensitivity of NISH for viral detection in this system is complex because of variation of signal size and number. Nevertheless, one signal per nucleus equates to as little as 30 to 40 viral copies, and probably much less. The ability to distinguish up to 15 discrete signals with both digoxigenin and biotin labelled probes in the nuclei of CaSki cells indicates that these methods will be useful in interphase cytogenetics in material routinely fixed in aldehyde.

Alkaline Phosphatase↗

PubMed1995

Quantitation of Chlamydia trachomatis by culture, direct immunofluorescence and competitive polymerase chain reaction.

OBJECTIVES: Methods to quantitate Chlamydia trachomatis have never been compared although it would be relevant to periodically evaluate the sensitivity of a detection system. We compared the sensitivity and reproducibility of culture, direct immunofluorescence and the polymerase chain reaction (PCR) to quantitate C trachomatis. METHODS: A competitive semiquantitative PCR procedure was developed. The number of inclusions in culture, particles by direct immunofluorescence and DNA copies by PCR were measured for 12 patient specimens. Variation was determined by measuring a sample 10 times for each method. RESULTS: Patient C trachomatis major outer membrane protein gene DNA was measured semiquantitatively by amplifying together with reference DNA. DNA molecules, particles and infectious units were quantitated in clinical samples with, on average, 595 DNA molecules and 87 immunofluorescent particles observed per inclusion-forming-unit. Similar coefficients of variation (47-52%) were observed for the 3 procedures. CONCLUSION: Competitive PCR and counting immunofluorescent particles provide reproducible and sensitive methods of quantitating C trachomatis.

Bacterial Outer Membrane Proteins↗

PubMed1996

Competitive RNA templates for detection and quantitation of growth factors, cytokines, extracellular matrix components and matrix metalloproteinases by RT-PCR.

Detection of low-abundance mRNAs by reverse transcription-polymerase chain reaction (RT-PCR) has become a standard technique to determine gene expression by tissues and cells in culture. The ability to determine relative or absolute copy number of specific mRNAs has been difficult due to inadequate internal standards to control for sample-to-sample variation. The use of a synthetic RNA standard with identical sequences to the PCR primers allows reproducible quantitation between samples and assays. By designing multi-sequence templates, several specific mRNAs can be quantitated using a single template. Addition of multiple templates to a single RT reaction allows the quantitation of a large number of targets from as little as 4 micrograms of total RNA. In this report, we present a series of seven primer/template systems to detect and quantitate 52 specific messages, including 26 growth factors and receptors, 8 extracellular matrix components, 10 matrix-modifying enzymes and their inhibitors and 8 cytokines.

Binding, Competitive↗

PubMed1996

Evolution of multiple families of non-LTR retrotransposons in phlebotomine sandflies.

In this paper we report on the diversity and distribution of a set of non-LTR retrotransposon (RTP) reverse transcriptase (RT) sequences isolated from phlebotomine sandflies, and their potential for investigating the evolutionary histories of members of this subfamily of flies (Diptera:Psychodidae, Phlebotominae). The phlebotomine RT sequence families derived from one species were as different from each other as they were from RT sequences derived from other species. When each was used to probe Southern blots of sandfly genomic DNA they hybridized only to the species of source and, usually, to others of the same subgenus, but not to DNA from other subgenera-a hybridization pattern consistent with vertical evolution. There was considerable intraspecific variation in hybridization pattern, suggesting the RTs were part of non-LTR RTPs that are (or were recently) subject to flux in genomic position and copy number. Most of the RT families detected in phlebotomines are monophyletic with respect to previously described RTs, and all are monophyletic with RTs of the F/Jockey (Drosophila melanogaster) type of RTP. Orthologous sequences were isolated from the closely related species Phlebotomus perniciosus and P. tobbi (subgenus Larroussius), and different populations of P. perniciosus. The level of sequence divergence among these orthologous RTs, the subgeneric distribution of each RT family, and the intraspecific variation in hybridization pattern of many of them, indicate this class of sequence will provide genetic markers at the sub-generic level.

Amino Acid Sequence↗

PubMed2006

Retrotransposon sequence variation in four asexual plant species.

Transposable elements (TEs) can be viewed as genetic parasites that persist in populations due to their capacity for increase in copy number and the inefficacy of selection against them. A corollary of this hypothesis is that TEs are more likely to spread within sexual populations and be eliminated or inactivated within asexual populations. While previous work with animals has shown that asexual taxa may contain less TE diversity than sexual taxa, comparable work with plants has been lacking. Here we report the results of a study of Ty1/copia, Ty3/gypsy, and LINE-like retroelement diversity in four asexual plant species. Retroelement-like sequences, with a high degree of conservation both within and between species, were isolated from all four species. The sequences correspond to several previously annotated retroelement subfamilies. They also exhibit a pattern of nucleotide substitution characterized by an excess of synonymous substitutions, suggestive of a history of purifying selection. These findings were compared with retroelement sequence evolution in sexual plant taxa. One likely explanation for the discovery of conserved TE sequences in the genomes of these asexual taxa is simply that asexuality within these taxa evolved relatively recently, such that the loss and breakdown of TEs is not yet detectable through analysis of sequence diversity. This explanation is examined by conducting stochastic simulation of TE evolution and by using published information to infer rough estimates of the ages of asexual taxa.

Amino Acid Sequence↗

PubMed2002

Transposable elements and the evolution of genome size in eukaryotes.

It is generally accepted that the wide variation in genome size observed among eukaryotic species is more closely correlated with the amount of repetitive DNA than with the number of coding genes. Major types of repetitive DNA include transposable elements, satellite DNAs, simple sequences and tandem repeats, but reliable estimates of the relative contributions of these various types to total genome size have been hard to obtain. With the advent of genome sequencing, such information is starting to become available, but no firm conclusions can yet be made from the limited data currently available. Here, the ways in which transposable elements contribute both directly and indirectly to genome size variation are explored. Limited evidence is provided to support the existence of an approximately linear relationship between total transposable element DNA and genome size. Copy numbers per family are low and globally constrained in small genomes, but vary widely in large genomes. Thus, the partial release of transposable element copy number constraints appears to be a major characteristic of large genomes.

DNA Transposable Elements↗

PubMed2026

Pan-genomics and multi-omics for deciphering genetic variation and accelerating genetic improvement in ruminant livestock.

Livestock reference genomes have transformed the discovery of variants associated with production, reproduction, health, and environmental adaptation. Nevertheless, a single linear reference represents only one mosaic haplotype and incompletely captures sequence diversity within a species, particularly structural variants, copy-number changes, repeat-rich regions, and breed-specific sequences. Pangenomes address this limitation by integrating multiple high-quality assemblies or population-scale variants into a unified sequence or graph representation. Concurrently, multi-omics approaches connect genomic variation with transcriptomic, epigenomic, manuscriptproteomic, metabolomic, and microbiome responses, thereby improving biological interpretation of genotype-phenotype relationships. This review synthesizes recent progress in livestock pangenomics and multi-omics, with emphasis on cattle, goats, sheep, water buffalo, and chickens. It describes advances in long-read and haplotype-resolved sequencing, graph construction, structural-variant discovery and genotyping, functional annotation, and integrative analysis. Recent pangenome studies have uncovered substantial non-reference sequence, reduced reference bias, identified breed- and population-specific structural variants, and resolved candidate variants underlying pigmentation, body size, tail morphology, cashmere production, altitude adaptation, and other economically relevant traits. However, translation into routine breeding remains constrained by uneven population representation, inconsistent structural-variant definitions, limited functional annotation, computational demands, and insufficient validation across environments. Future progress will depend on diverse near-complete assemblies, graph-aware imputation and genomic prediction, long-read transcriptomics, single-cell and spatial omics, rigorous causal validation, and open, interoperable resources. Together, these developments can support more accurate, resilient, and biologically informed livestock improvement. Importantly, current dairy-cattle evidence indicates that pangenome-derived structural variants can substantially improve variant discovery and functional interpretation while yielding only marginal average gains in routine genomic prediction, favoring targeted augmentation rather than wholesale replacement of established SNP-based evaluations.

Animals↗