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PubMed2000

Mitochondrial genome variation and the origin of modern humans.

The analysis of mitochondrial DNA (mtDNA) has been a potent tool in our understanding of human evolution, owing to characteristics such as high copy number, apparent lack of recombination, high substitution rate and maternal mode of inheritance. However, almost all studies of human evolution based on mtDNA sequencing have been confined to the control region, which constitutes less than 7% of the mitochondrial genome. These studies are complicated by the extreme variation in substitution rate between sites, and the consequence of parallel mutations causing difficulties in the estimation of genetic distance and making phylogenetic inferences questionable. Most comprehensive studies of the human mitochondrial molecule have been carried out through restriction-fragment length polymorphism analysis, providing data that are ill suited to estimations of mutation rate and therefore the timing of evolutionary events. Here, to improve the information obtained from the mitochondrial molecule for studies of human evolution, we describe the global mtDNA diversity in humans based on analyses of the complete mtDNA sequence of 53 humans of diverse origins. Our mtDNA data, in comparison with those of a parallel study of the Xq13.3 region in the same individuals, provide a concurrent view on human evolution with respect to the age of modern humans.

Africa↗

PubMed1975

Localization of rDNA and Giemsa-banded chromosome complement of white-handed gibbon, Hylobates lar.

A karyotype based on banding pattern and chromosome length is presented for the white-handed gibbon, Hylobates lar. Little homology with the banding patterns of the chromosomes of the other Hominoidea can be seen, confirming the early evolutionary separation of Hylobatidae and the other apes. Hybridization in situ with ribosomal RNA shows that the secondary constriction of a submetacentric chromosome (15) is the only site of the nucleolar organizer, as in the Cercopithecoidea. The correlation of polymorphic variation in size of this secondary constriction with grain density suggests differences in the number of gene copies per chromosome.

Animals↗

PubMed1976

DNA reassociation kinetics in relation to genome size in four amphibian species.

DNA reassociation kinetics were studied, by means of the hydroxyapatite chromatography method, for four species of Amphibians with different nuclear DNA content: Xenopus laevis (3 pg DNA per haploid genome) and Bufo bufo (7 pg) of the Anura subclass and Triturus cristatus (23 pg) and Necturus maculosus (52 pg) of the Urodela subclass. Within each subclass the two species studied were found to have about the same absolute amount of unique DNA. The differences of total nuclear DNA can be accounted for by quantitative variations of the repetitive sequence classes, at least in part due to changes in the number of copies of the various sequences. On the contrary the great difference in nuclear DNA between the two subclasses, Anura and Urodela, involves all sequence classes in parallel; the slowly reassociating fraction appears to be unique in spite of a tenfold difference in absolute amount. The dependence of reassociation kinetics on DNA fragment length for the four species indicates for all of them an interspersed organization of the various sequence classes.

Amphibians↗

PubMed2003

A single nucleotide polymorphism in the Emp3 gene defines the H4 minor histocompatibility antigen.

Minor histocompatibility antigens (minor H antigen) elicit strong T-cell-mediated responses during both graft rejection and graft versus leukemia (GvL) among MHC-matched individuals (where MHC is major histocompatibility complex). Employing expression-cloning methodology, we have identified a cDNA clone, MI-35, encoding the immunodominant H4b minor H antigen within the classical mouse H4 complex. The minimal antigenic epitope derived from H4b presented on Kb class I MHC is SGIVYIHL (SYL8) and the polymorphism is due to C-->T nucleotide modification in p3 resulting in the change of threonine (ACT) to isoleucine (ATT). The results presented here demonstrate that amino acid variation in the allelic epitopes results in the low abundance of H4a peptide. The differential peptide copy number resulted in an immunodominant cytotoxic T cells (CTL) response directed against H4b while the anti-B6 response directed against H4a was easily dominated. These results provide a molecular mechanism for the H4 minor H antigen and suggest a novel mechanism by which alloantigenic disparity caused by conservative amino acid changes can be augmented by posttranslational antigen processing events.

Amino Acid Sequence↗

PubMed2005

Chromosomal alterations detected by comparative genomic hybridization in nonfunctioning endocrine pancreatic tumors.

We have used comparative genomic hybridization to investigate changes in DNA copy number in 20 nonfunctioning endocrine pancreatic tumors. The total number of regional DNA imbalances per tumor showed variation from case to case and high genetic heterogeneity was observed. From 1 to 22 chromosomal anomalies were detected in 13 of the 20 cases evaluated. Overall gains predominated over losses with a ratio of about 3.9:1 (58 gains/15 losses). The mean total number of regions displaying imbalances increased from 1.25 per tumor for benign tumors to 5.25 for malignant tumors, although statistical significance was not reached (P=0.074).

Adult↗

PubMed2003

Noise in gene expression as the source of non-genetic individuality in the chemotactic response of Escherichia coli.

A deterministic computer model of the signal transduction pathway mediating bacterial chemotaxis was used to examine the variation in both unstimulated swimming behaviour and adaptation time to stimuli in clonal populations of cells. Copy numbers of proteins in the pathway were computed from a simplified model of transcription and translation that predicts greater-than-Poissonian statistics. Simulated and experimental individuality data could be brought into good agreement on varying the noise strength of the protein copy number distributions. In the simulations, all the proteins in the pathway are involved to a significant degree in the appearance of phenotypic diversity, although there is a modest decrease in influence with increasing copy number.

Adaptation, Biological↗

PubMed2001

Energy transfer between fluorescent proteins using a co-expression system in Mycobacterium smegmatis.

The goal of this study was to establish a two-plasmid co-expression system for Mycobacterium smegmatis. Two vectors with compatible origins of replication and a polylinker, which allows modular cloning of promoters and genes, were constructed and used to clone genes encoding a blue fluorescent protein (BFP) and a green fluorescent protein (GFP). A 160-fold variation of GFP expression levels in M. smegmatis was achieved by combining three promoters with different copy numbers of the vectors. An efficient energy transfer between BFP and GFP in M. smegmatis was observed by fluorescence measurements and demonstrated that these genes were simultaneously expressed from both vectors. Thus, these vectors will be valuable for all strategies where co-expression of proteins in M. smegmatis is needed, e.g. for constructing a two-hybrid system or for deleting essential genes.

Base Sequence↗

PubMed1997

PCR analysis of human telomeric repeats present on HHV-6A viral strains.

Human herpesvirus 6 (HHV-6) presents a perfect tandem array of human telomeric repeats (TRS) at both identical direct repeats (DR). Several researchers have reported a different TRS copy number by sequence analysis of HHV-6 DR's cloned fragments so it has been hypothesized that number of TRS is unstable. By PCR we show that the TRS copy number of U1102 HHV-6 variant A strains is stable during viral cultivation in cell lines and each HHV-6 variant A strain, detected in pathologic specimens, is characterized by a specific TRS copy number.

Cell Line↗

PubMed2002

Quantification of substance p mRNA in human immune cells by real-time reverse transcriptase PCR assay.

We have applied a newly developed real-time reverse transcriptase (RT) PCR (RT-PCR) assay for quantification of substance P (SP) mRNA expression (the SP real-time RT-PCR assay) in human blood monocyte-derived macrophages, peripheral blood lymphocytes, and microglia isolated from fetal brain. The SP real-time RT-PCR assay had a sensitivity of 60 mRNA copies, with a dynamic range of detection between 60 and 600,000 copies of the SP gene transcript per reaction mixture. The coefficient of variation of the threshold cycle number between the SP real-time RT-PCR assays was less than 1.16%. This assay with an SP-specific primer pair efficiently recognizes all four isoforms of preprotachykinin A (the SP precursor) gene transcripts. In order to use this assay to measure the levels of SP mRNA in the human immune cells quantitatively, we designed a specific probe (molecular beacon) derived from exon 3 of the SP gene. We demonstrated that the real-time RT-PCR quantitatively detected SP mRNA in the human immune cells, among which the microglia isolated from fetal brain had the highest levels of SP mRNA. The SP real-time PCR assay yielded reproducible data, as the intra-assay variation was less than 1%. Thus, it is feasible to apply the real-time RT-PCR assay for quantification of SP mRNA levels in human immune cells, as well as in other nonneuronal cells. Since SP is a major modulator of neuroimmunoregulation, this assay has the potential for widespread application for basic and clinical investigations.

Adult↗

PubMed1990

The chicken lysozyme 5' matrix attachment region increases transcription from a heterologous promoter in heterologous cells and dampens position effects on the expression of transfected genes.

Matrix attachment regions (MARs) are DNA elements that dissect the genome into topologically separated domains by binding to a chromosomal skeleton. This study explored the putative influence of the MAR located 5' of the chicken lysozyme gene on expression of heterologous genes in heterologous cell systems. Expression of a construct with the chloramphenicol acetyltransferase (CAT) indicator gene controlled by the herpes simplex virus thymidine kinase promoter (TC) and a construct in which the same transcriptional unit is flanked by chicken lysozyme 5' MARs (MTCM) was assayed after stable transfection into rat fibroblasts. Median CAT activity per copy number in MTCM transfectants was elevated approximately 10-fold relative to that in TC transfectants. Total variation in normalized CAT activity decreased from more than 100-fold among TC transfectants to nearly 6-fold among MTCM transfectants. The steady-state level of transcripts and the relative rate of transcription were increased in MTCM transfectants, as shown by S1 nuclease and run-on transcription assays, respectively. The chicken lysozyme 5' MAR thus can confer elevated, less position-dependent expression on a heterologous promoter in cells of a different species by increasing the density of transcribing RNA polymerase molecules. MAR-mediated transcriptional enhancement suggests that MARs are important for gene expression and not just for DNA packaging.

Animals↗

PubMedDate not supplied

Complex structure of knobs and centromeric regions in maize chromosomes.

The recovery of maize (Zea mays L.) chromosome addition lines of oat (Avena sativa L.) from oat x maize crosses enables us to analyze the structure and composition of individual maize chromosomes via the isolation and characterization of chromosome-specific cosmid clones. Restriction fragment fingerprinting, sequencing, and in situ hybridization were applied to discover a new family of knob associated tandem repeats, the TR1, which are capable of forming fold-back DNA segments, as well as a new family of centromeric tandem repeats, CentC. Analysis of knob and centromeric DNA segments revealed a complex organization in which blocks of tandemly arranged repeating units are interrupted by insertions of other repeated DNA sequences, mostly represented by individual full size copies of retrotransposable elements. There is an obvious preference for the integration/association of certain retrotransposable elements into knobs or centromere regions as well as for integration of retrotransposable elements into certain sites (hot spots) of the 180-bp repeat. DNA hybridization to a blot panel of eight individual maize chromosome addition lines revealed that CentC, TR1, and 180-bp tandem repeats are found in each of these maize chromosomes, but the copy number of each can vary significantly from about 100 to 25,000. In situ hybridization revealed variation among the maize chromosomes in the size of centromeric tandem repeats as well as in the size and composition of knob regions. It was found that knobs may be composed of either 180-bp or TR1, or both repeats, and in addition to large knobs these repeated elements may form micro clusters which are detectable only with the help of in situ hybridization. The association of the fold-back elements with knobs, knob polymorphism and complex structure suggest that maize knob may be consider as megatransposable elements. The discovery of the interspersion of retrotransposable elements among blocks of tandem repeats in maize and some other organisms suggests that this pattern may be basic to heterochromatin organization for eukaryotes.

Avena↗

PubMed1991

Semiquantitative analysis of integrated genomes of human T-lymphotropic virus type I in asymptomatic virus carriers.

A semiquantitative estimation of human T-lymphotropic virus type I (HTLV-I) integration by peripheral blood mononuclear cells (PBMC) was performed. Genomic DNA samples derived from 134 HTLV-I carriers were subjected to 40 or 60 cycles of the polymerase chain reaction to amplify the pol region of HTLV-I. The HTLV-I genome was detected by dot hybridization using a 32P-labeled oligonucleotide probe for the pol region. The radioactivity of hybridized dot membranes was then counted with an RI Imaging System (Ambis Inc, San Diego, CA) and the HTLV-I genome dose was determined by comparison with standard curve for serially diluted HTLV-I genome-positive DNA. A wide range of variation of HTLV-I genome integration was observed. When the integrated genome dose was calculated as the number of HTLV-I copies per 100 PBMC, 7 carriers (5%) had more than 10 copies, 56 (42%) had 1 to 10 copies, 46 (34%) had 0.1 to 1 copy, and 24 (18%) had less than 0.1 copy. In one sample, the HTLV-I genome was undetectable, which may indicate that the integrated genome was present at less than 0.01 copies per 100 PBMC. Age- or sex-related variations in the distribution of individuals with different HTLV-I genome were rather limited. However, carriers with a high level of the HTLV-I genome were always more than 30 years old and were predominantly male (six of seven).

Aging↗

PubMed2006

Structural variation in the human genome.

The first wave of information from the analysis of the human genome revealed SNPs to be the main source of genetic and phenotypic human variation. However, the advent of genome-scanning technologies has now uncovered an unexpectedly large extent of what we term 'structural variation' in the human genome. This comprises microscopic and, more commonly, submicroscopic variants, which include deletions, duplications and large-scale copy-number variants - collectively termed copy-number variants or copy-number polymorphisms - as well as insertions, inversions and translocations. Rapidly accumulating evidence indicates that structural variants can comprise millions of nucleotides of heterogeneity within every genome, and are likely to make an important contribution to human diversity and disease susceptibility.

Genetic Variation↗

PubMed1981

Restriction enzyme analysis of the germ line limited DNA of Ascaris suum.

The germ line limited DNA of Ascaris suum was isolated from sperm and testis as a satellite DNA component in Hoechst 33 258 -- CsCl gradients. Employing restriction enzyme analysis, we show that the germ line limited DNA is composed entirely of two families of tandemly repeated sequences, one repeat unit is 125 bp, and the other 131 bp long. The total appr. 5 x 10(5) copies of the two families are physically separated from each other (segmental arrangement). Several repeat unit variants within both families could be detected. The copies of sequence variants are arranged in tandem (subsegmental arrangement). Reassociation and hybridization experiments revealed similar sequences of the two repeat units. The archaeotypic core sequence of both repeat units is probably a tetranucleotide which shows a 'theme and variation' pattern. During chromatin diminution in the presoma cells the satellite DNA is eliminated from the chromosomes. However, a limited number of tandemly repeated copies of both kinds of repeat units could be detected in the soma genome using radioactive probes of both repeat units in Southern blots of muscle and intestine of adult animals. The tandem arrangement and the hierarchical pattern of restriction sites throughout different subfamilies supports the model of successive segmental amplification events during the evolution of the germ line limited DNA. Since the germ line limited satellite DNA is exclusively located at the ends of the chromosomes, a fold back structure for the telomeric DNA sequences is proposed which might have generated this DNA.

Animals↗

PubMed2001

An insulator element from the sea urchin Hemicentrotus pulcherrimus suppresses variation in transgene expression in cultured tobacco cells.

Specialized DNA sequences known as insulators protect genes from both the positive and negative influences of nearby chromatin. Many insulators have been identified in various species; however, few function in multiple species. We have shown that an insulator from the Ars (arylsulfatase) gene of the sea urchin Hemicentrotus pulcherrimus functions in plant cells. Normally, expression of an introduced chimeric GUS gene is inactivated in approximately 30% of transformed tobacco BY2 clones. Transgenes containing the Ars insulator, however, were expressed in all transformed tobacco BY2 cells. The insulator did not affect the copy number, the chromosomal position of transgene integration or maximum expression levels. These results suggest that the insulator functions to suppress the variation normally associated with transgene expression in tobacco BY2 cells.

Animals↗

PubMedDate not supplied

A 1.3 kb satellite DNA from Bubalus bubalis not conserved evolutionarily is transcribed.

A 1.3 kb satellite DNA from a size defined genomic library of mammal Bubalus bubalis was cloned and sequenced. The clone pSB1 is AT rich with 447 A (33.6%), 262 C (19.7%), 240 G (19.0%) and 383 T (28.8%). There were about 1400 copies of contig in the bubaline genome but it did not uncover allele length variation when used as probe in conjunction with a number of restriction enzymes. The contig pSB1 is not conserved evolutionarily and cross hybridizes only with the Bovideae family. A set of primers from 5' (nt 422 to 441) and 3' (nt 962 to 947) deduced from the clone used for PCR amplification with four members of the Bovideae family gave the expected 530 bp band of equal intensity indicating a similar number of copies in all the four species namely Bos indicus, Capra hircus, Ovis aries and Bubalus bubalis. Expression studies with pSB1 following slot-blot hybridization with total RNA isolated from ovary, testes, kidney, lung and spleen revealed varying signal intensities in all the tissues with a most prominent signal in spleen but a faint one in ovary. Further sequence analysis revealed the presence of several eukaryotic transcriptional elements such as NF-E1, Poly-A signal, lariat consensus sequences, and CTF/NF1 binding sites. Blast search showed 90% sequence similarity with the reverse transcriptase gene of Bos taurus and sequences from nt 283 to 636 within the contig showed highly conserved reverse transcriptase like signatures along with N-glycosylation and protein kinase C phosphorylation sites. From the data we conclude that the pSB1 representing satellite DNA is associated with transcribing sequences. The prospect of identifying functional genes linked with the satellite fraction in higher vertebrates is discussed.

Animals↗

PubMed2000

Primary human bone cultures from older patients do not respond at continuum levels of in vivo strain magnitudes.

Osteoporosis is characterized by excessive loss of bone mass, while exercise is believed to maintain or enhance bone mass. Since exercise marginally affects osteoporosis, we wondered whether bone cells from osteoporotic patients would fail to respond to strain. Primary human bone-like cultures were obtained from females over age 60 with hip arthroplasty procedures performed for either osteoporotic fracture (n = 8) or non-osteoporotic osteoarthrosis (n = 5). Cultures (96,000 cell/cm2) were strained in rectangular optically clear silastic wells. Three periods of uniaxial substratum strain (1000 micro-strain, 1 Hz, 10,000 cycles, sine wave) were provided every 24 h using a four-point bending, computer-controlled device. Results at a frequency of 1 Hz were compared to cultures exposed to 20 Hz with bone cells derived from one osteoarthritic subject. Alterations in protein level expression of bone-related proteins were determined using a semi-quantitative confocal approach along with enzyme (alkaline phosphatase) activity and enzyme mRNA copy number using cRNA RT-PCR. Strain did not alter levels of bone-related protein levels, enzyme activity, or steady state copy number per cell in response to strain in either group. Strained cultures from osteoporotic patients exhibited little variation from unstrained controls, while individual cultures from osteoarthritic patients exhibited increases in one protein or the other. The results suggest that bone cells from older individuals may not be responsive to continuum levels of strain anticipated with vigorous activity.

Aged↗

PubMed2003

Analysis of cell-specificity and variegation of transgene expression driven by salmon prolactin promoter in stable lines of transgenic rainbow trout.

In order to identify the specificity and functionality of salmon prolactin (sPRL) promoter, transgenic rainbow trout carrying a construct comprising the 2.4 kb fragment of the 5' flanking region of Atlantic Chinook sPRL gene fused either to the reporter genes cat (sPRL-cat) or lacZ (sPRL-lacZ) were produced. sPRL-cat in transgenic F0 fish expressed strongly CAT only in the pituitary gland. Transgenic in F1-F4 lines harbouring sPRL-lacZ expressed beta-galactosidase (beta-gal) only in the follicular PRL-producing cells of the adenohypophysis. We observed heterocellular, mosaic distribution of beta-gal within PRL cell population and enormous variation of lacZ expression level between the littermates in the same transgenic line. Regardless of the transgene copy number, age or sex of transgenic fish, beta-gal expression was lactotroph-specific but variegated in all the nine F2 hemizygous lines analysed. One line harbouring a multicopy integration was followed up to F4 generation: the transgene was transmitted without modifications. Analysis of genomic DNA from pituitaries showed that lacZ sequences were highly methylated. LacZ expression was low and its transcripts, analysed by in situ hybridisation, showed a mosaic distribution within the pituitary gland. These data suggest that variegated expression of lacZ can occur at the transcription level owing to the silencing effect of lacZ gene. After proving the tissue-specific expression of reporter genes driven by the sPRL promoter, we tried to obtain the genetic ablation of PRL-producing cells,by transferring the same construct comprising diphtheria toxin DT-A gene (tox). However, the high mortality rate of sPRL-tox transformed embryos has embedded this study and no transgenic fish expressing tox were produced. The appropriateness of using transgenic strategies to analyse gene function in Salmonids is discussed, especially the implications of the multicopy integration patterns and of the variegated transgene expression.

Animals↗