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PubMed1989

Patterns of intra- and interarray sequence variation in alpha satellite from the human X chromosome: evidence for short-range homogenization of tandemly repeated DNA sequences.

A number of processes, such as sequence conversion, unequal crossingover, and molecular drive, have been postulated to explain the homogenization of tandemly repeated DNA families. To investigate the nature and extent of such processes in the alpha satellite family of centromeric DNA, we determined the nucleotide sequence of approximately 700 bp from each of 40 representative alpha satellite repeats from six sources of human X chromosomes, obtaining a total of approximately 28 kb of sequence data. Sequence divergence among the repeats examined was low, with an average pairwise difference of approximately 1%. Pairwise comparisons of all repeats indicate that the degree of similarity for those repeats in physical proximity (within approximately 15 kb) of each other is significantly greater than that for randomly located repeats, from either the same or different X chromosomes, suggesting that the mechanisms predicted to homogenize these arrays are effectively short-range in action. Analysis of individual patterns of sequence variation allows the assignment of haplotypes for five high-copy-number diagnostic positions and reveals distinct positions of equilibrium and disequilibrium within the repeat. These analyses address hypotheses about the origin of the observed patterns of variation throughout alpha satellite evolution.

Base Sequence↗

PubMed2006

Screening of genetic and expression alterations of SRC1 gene in prostate cancer.

BACKGROUND: Genetic alterations of the SRC1 gene have not been thoroughly studied in prostate cancer. MATERIALS AND METHODS: Five prostate cancer cell lines and 32 xenografts were screened for mutations and gene copy number alterations. Subsequently, frequencies of detected sequence variations were further analyzed in 44 clinical prostate cancers, 6 benign prostate hyperplasias, and 48 normal controls. Finally, the protein expression of SRC1 in 254 clinical prostate tumors was investigated. RESULTS: Three non-recurrent sequence variations, and one single nucleotide polymorphism in the coding region of SRC1, as well as one case of SRC1 gene amplification were found. The protein expression of SRC1 was higher in androgen ablation resistant than untreated prostate carcinomas, but the difference was not statistically significant (P = 0.0796). CONCLUSIONS: Genetic alterations of SRC1 are rare in prostate cancer. The nuclear protein accumulation of SRC1 seems to be mildly increased in androgen ablation resistant prostate cancers. .

Androgen Antagonists↗

PubMed1994

Structure and possible biological roles of Lp(a).

Lipoprotein(a) [Lp(a)] is a plasma macromolecular complex that is assembled from low-density lipoproteins (LDL) and a large hydrophilic glycoprotein, named apolipoprotein(a) [apo(a)], linked by a disulfide bond to apolipoprotein B-100. Apo(a) is formed by different structural domains one of which is present in multiple copies, the number of which is determined by variation in the hypervariable apo(a) gene. Sequence homology of apo(a) with plasminogen may explain the competition of Lp(a) for some physiological functions of plasminogen in the coagulation and fibrinolytic cascade in vitro. There is evidence that high plasma levels of Lp(a) may have atherogenic and/or thrombogenic potential. More work will have to be done to understand the exact role of Lp(a) in atherogenesis, to evaluate the potential synergy between Lp(a) and LDL in promoting coronary artery disease, and to assess the therapeutic benefits of a reduction of Lp(a) levels.

Biological Evolution↗

PubMed2001

Structure-function relationships of complement receptor type 1.

Human complement receptor type 1 (CR1) is a large, multifunctional glycoprotein which is a member of the regulators of complement activation family. Like other members of this family, it is composed mainly of tandemly arranged modules, each about 60-70 amino acids long, known as complement control protein repeats (CCPs). Each domain folds independently and contains a hydrophobic core wrapped in beta sheets. These domains mediate interactions with C3/C4-derived fragments. CR1 is the most versatile inhibitor of both classical and alternative pathway C3 and C5 convertases due to its decay-accelerating activity and co-factor activity for C3b/C4b cleavage. Moreover, CR1 plays a major role in immune complex clearance due to its high affinity for C3b and C4b. CR1 is an excellent model to study structure-function relationships because its functions are mediated by two distinct but highly homologous sites, each composed of three CCPs. CR1 derivatives carrying just one active site were used to define critical sequences/amino acids. This was achieved by testing functional profiles of the proteins carrying a mutated active site produced by substituting peptides/amino acids with their counterparts from the other site. These mutated proteins, of which we analyzed over 100, permitted the fine mapping of the functional sites. CR1 on primate erythrocytes varies in size. In most cases it is smaller and has fewer active sites than does human CR1. This variation was used to determine that increased copy number (3,000 to 20,000 versus 300 for human CR1) compensates for a smaller size. Moreover, studies of primate CR1 led to the finding that subtle differences in the critical areas, as compared to human sites, produce active sites with a broader functional repertoire. These alterations ensure that short CR1 forms possess similar biologic activities to the large CR1 forms. There is much interest in producing therapeutic agents to inhibit unwanted complement activation. Based on these structure-function analyses, smaller and more potent complement inhibitors derived from CR1 can be produced.

Amino Acid Sequence↗

PubMed2003

Quantitative real-time PCR based on single copy gene sequence for detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis.

OBJECTIVE: To establish a method for quantification of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis from subgingival plaque by real-time polymerase chain reaction (PCR) technique. MATERIAL AND METHODS: Bacterial cells from both species were obtained from type culture and counted microscopically. Cellular suspension in sterile distilled water was used for DNA extraction by boiling for 20 min, with a mineral oil cover. Primers for PCR were selected from sequences of LktC gene (A. actinomycetemcomitans) and Arg-gingipain (P. gingivalis) to yield amplicons below 100 bp. SYBR Green I based real-time PCR was adjusted to quantify separately both species. RESULTS: A good sensitivity and specificity were obtained for both species, although the yield was better for A. actinomycetemcomitans. A good repeatability of cycle threshold (CT) was encountered, so coefficient of variation was below 6% at every initial copy number. CONCLUSION: A new method of quantification of A. actinomycetemcomitans and P. gingivalis based on SYBR Green real-time PCR is presented. Its good sensibility and repeatability will allow its application to analysis of subgingival plaque samples.

Aggregatibacter actinomycetemcomitans↗

PubMed2001

Positive selection of a gene family during the emergence of humans and African apes.

Gene duplication followed by adaptive evolution is one of the primary forces for the emergence of new gene function. Here we describe the recent proliferation, transposition and selection of a 20-kilobase (kb) duplicated segment throughout 15 Mb of the short arm of human chromosome 16. The dispersal of this segment was accompanied by considerable variation in chromosomal-map location and copy number among hominoid species. In humans, we identified a gene family (morpheus) within the duplicated segment. Comparison of putative protein-encoding exons revealed the most extreme case of positive selection among hominoids. The major episode of enhanced amino-acid replacement occurred after the separation of human and great-ape lineages from the orangutan. Positive selection continued to alter amino-acid composition after the divergence of human and chimpanzee lineages. The rapidity and bias for amino-acid-altering nucleotide changes suggest adaptive evolution of the morpheus gene family during the emergence of humans and African apes. Moreover, some genes emerge and evolve very rapidly, generating copies that bear little similarity to their ancestral precursors. Consequently, a small fraction of human genes may not possess discernible orthologues within the genomes of model organisms.

Animals↗

PubMed2006

Differences in vertebrate microRNA expression.

MicroRNAs (miRNAs) attenuate gene expression by means of translational inhibition and mRNA degradation. They are abundant, highly conserved, and predicted to regulate a large number of transcripts. Several hundred miRNA classes are known, and many are associated with cell proliferation and differentiation. Many exhibit tissue-specific expression, which aids in evaluating their functions, and it has been assumed that their high level of sequence conservation implies a high level of expression conservation. A limited amount of data supports this, although discrepancies do exist. By comparing the expression of approximately 100 miRNAs in medaka and chicken with existing data for zebrafish and mouse, we conclude that the timing and location of miRNA expression is not strictly conserved. In some instances, differences in expression are associated with changes in miRNA copy number, genomic context, or both between species. Variation in miRNA expression is more pronounced the greater the differences in physiology, and it is enticing to speculate that changes in miRNA expression may play a role in shaping the physiological differences produced during animal development.

Animals↗

PubMed1989

Genotypic and phenotypic evidence of clonal interactions in murine tumor cells.

The stability of mixed tumor cell populations has been described in terms of phenotypic characteristics such as metastatic potential, immunogenicity, and drug resistance. We have extended these analyses to the molecular level in a model that uses transfection of the hemagglutinin antigen (HA) gene of influenza virus into murine CT-26 colorectal carcinoma cells. Transfection was followed by fluorescence-activated cell sorting (FACS) to select a parent population with expression of high levels of HA. We characterized this population (FACS-3) and four derived clones (5, 6, 9, and 18) over time with regard to phenotypic characteristics: immunogenicity and cross-protection against tumor challenge, cell surface expression of HA, evidence of HA gene amplification, and levels of HA mRNA. During 6 months in culture, the FACS-3 parent cells remained stable, but the individual clones varied for all of the parameters assessed. Among the clones, all possible molecular variations occurred, including changes in HA gene copy number (increased in clone 5 and decreased in clone 18), gene rearrangement (clone 5), decrease in HA mRNA (clones 6, 9, and 18), increase in HA mRNA (clone 5), and an abnormality in translational control or a posttranslational error. In all cases, the molecular changes correlated with cell surface HA expression, immunogenicity, and cross-protective potential. We conclude that in vitro clonal interactions play a role in stabilizing heterogeneity in this system. These studies show that even in the absence of selection, clonal interactions may alter the phenotype of tumors by increasing malignant progression or impeding tumor growth.

Animals↗

PubMed1994

Depletion of hepatitis C virus from procured kidneys using pulsatile perfusion preservation.

The safety of the use of kidneys procured from a hepatitis C virus (HCV)-positive cadaver donors in transplantation has recently been the subject of controversy. One factor that is important in determining the transmission of the virus and/or viral liver disease is the total viral inoculum to which the renal allograft recipient is exposed as a result of the transplant. We have studied the effect of a standard pulsatile renal preservation procedure and variations of it on the number of viral copies in organs from HCV-positive donors. An HCV-RNA quantitative reverse transcription-polymerase chain reaction (RT-PCR) method was utilized with a recombinant competitive inhibitor substrate added after cDNA synthesis with the PCR primers within the relatively non-polymorphic 5' untranslated region of the HCV genome. Additionally, strain specificity was found to be detectable using a modification of the technique of restriction fragment-length polymorphism (RFLP-PCR), so that virus from the organ donor could be specifically identified and quantified in the recipient. It was observed that standard preservation procedures using pulsatile perfusion were able to eliminate 75% of the virus from the organ in 20 hr. By modifying this procedure to include additional wash steps and a second pulsatile perfusion, greater than 99% of the virus could be eliminated from the kidney. In a related study, we used quantitative PCR to study requirements for filtration of the virus using HCV-positive serum. It was found that a high-flow-rate ultrafilter with a molecular weight cut-off (MWCO) of 300,000 daltons placed in series to the preservation apparatus was very efficient in eliminating the virus from perfusion solution in less than 2 hr. It can therefore be proposed that with the use of these molecular techniques, pulsatile perfusion coupled with additional viral depletion steps (dilution, and/or filtration) may allow the practical reduction of HCV transmission risk in recipient follow-up studies. The means are thereby presented for similar manipulation of other known or, as yet, unknown transmissible agents.

Base Sequence↗

PubMed2003

[Effect of learning about the human genome on the development of pathology].

On the basis of data of the Human Genome Project, it was embraced the newest information about the gene content of the human genome, the disease genes, the parasitic DNA, the single nucleotide polymorphisms, the repeat sequences, the cytoskeletons, the regulation of cell proliferation, and their medical consequences. The applicability of the acquaintance with the human genome in pathology is presented with a few examples of our own. The significance of the single nucleotide polymorphisms in susceptibility to, or protection from, a host of disease is illustrated by the example of the allele variation of Apo-E gene. The copy number of the N-myc gene in neuroblastomas and HER2/neu gene in breast carcinomas was determined with quantitative PCR techniques. The monoclonally increased abnormal p53 protein expression was found in small cell lung cancer (in 90% frequency), in oro-pharyngeal carcinomas (82%), in esophageal squamous cell carcinomas (59%) in stomach cancer (33%), in colon carcinomas (27%) and in soft tissue sarcomas (13%). These data advert to the fact that the mutation of the p53 gene is much more frequent in those tumors in which the basic tissue is directly exposed to with the environmental carcinogens. It is now known, that near the repetitive sequences, gene rearrangement can more easily be evolve. Finally, we have determined the conditions of the accomplishment of the molecular pathological diagnosis: (1) It is applicable, when the classic morphology does not eventuate a conclusive result. (2) Well known and validated gene alterations are admissible to diagnostic purpose. (3) Only standard methods are applicable along with positive and negative controls. (4) The result has to correlate with the morphological picture, the immunohistochemical profile and the clinical data. (5) It is necessary to be able to appropriately interpret the molecular biological result, which is then incorporated in the pathological report. (6) The ethical, legal and social consequences must be considered.

Cell Division↗

PubMed1988

Evidence for frequent gene conversion in the steroid 21-hydroxylase P-450(C21) gene: implications for steroid 21-hydroxylase deficiency.

Oligonucleotide probes specific for the deleterious mutations harbored in the P-450(C21)A pseudogene and oligonucleotide probes specific for the corresponding sequences in the B gene were prepared to examine the molecular lesions in the P-450(C21) gene of P-450(C21)-deficient patients. Using these gene-specific probes, we performed Southern blot analyses of genomic DNAs from 11 patients and eight normal individuals. At least one allele of the B gene (the 3.7-kb TaqI fragment) in a patient was inactivated by mutations caused by recombination with the A gene. The A genes in normal individuals and patients seemed to be replaced frequently (i.e., 10/19 individuals) in their 3' portions by B gene sequences. All of these alterations occurred without changing the characteristic length (3.2 kb) of the TaqI fragment of the A gene, a result strongly suggesting that frequent gene conversions and/or intragenic recombinations have happened in the P-450(C21) genes. Densitometric analysis of the autoradiograms from hybridization experiments revealed extensive variation (from one to five copies) in the copy number of the A gene (the 3.2-kb TaqI fragment) whereas that of the B gene (the 3.7-kb TaqI fragment) was relatively constant at two or three copies.

Adrenal Hyperplasia, Congenital↗

PubMed1990

copia expression is variable among natural populations of Drosophila.

A survey of copia (retroviral-like element) expression in flies representing 37 populations worldwide of Drosophila melanogaster, Drosophila simulans and Drosophila mauritiana demonstrates that, although copia elements are present in all three species, copia-encoded transcripts are detectable only in D. melanogaster. Levels of copia transcripts vary nearly 100-fold among flies representing geographically diverse populations of D. melanogaster and this variation is not correlated with variability in copia copy number. Analysis of transcript levels in interpopulation hybrids demonstrates that much of this variability may be attributable to the action of trans-acting controls. The geographic and phylogenetic pattern of copia expression suggests that moderate to high levels of copia expression may be a relatively recent evolutionary acquisition. The potential evolutionary significance of these findings is discussed.

Animals↗

PubMed1995

Cloning and characterization of the majority of repetitive DNA in cotton (Gossypium L.).

Repetitive DNA elements representing 60-70% of the total repetitive DNA in tetraploid cotton (Gossypium barbadense L.) and comprising 30-36% of the tetraploid cotton genome were isolated from a genomic library of DNA digested with a mixture of four blunt-end cutting restriction enzymes. A total of 313 clones putatively containing nuclear repetitive sequences were classified into 1103 families, based on cross hybridization and Southern blot analysis. The 103 families were characterized in terms of genome organization, methylation pattern, abundance, and DNA variation. As in many other eukaryotic genomes, interspersed repetitive elements are the most abundant class of repetitive DNA in the cotton genome. Paucity of tandem repeat families with high copy numbers (>10(4)) may be a unique feature of the cotton genome as compared with other higher plant genomes. Interspersed repeats tend to be methylated, while tandem repeats seem to be largely unmethylated in the cotton genome. Minimal variation in repertoire and overall copy number of repetitive DNA elements among different tetraploid cotton species is consistent with the hypothesis of a relatively recent origin of tetraploid cottons.

Base Sequence↗

PubMed2006

DNA copy-number analysis in bipolar disorder and schizophrenia reveals aberrations in genes involved in glutamate signaling.

Using bacterial artificial chromosome (BAC) array comparative genome hybridization (aCGH) at approximately 1.4 Mbp resolution, we screened post-mortem brain DNA from bipolar disorder cases, schizophrenia cases and control individuals (n=35 each) for DNA copy-number aberrations. DNA copy number is a largely unexplored source of human genetic variation that may contribute risk for complex disease. We report aberrations at four loci which were seen in affected but not control individuals, and which were verified by quantitative real-time PCR. These aberrant loci contained the genes encoding EFNA5, GLUR7, CACNG2 and AKAP5; all brain-expressed proteins with known or postulated roles in neuronal function, and three of which (GLUR7, CACNG2 and AKAP5) are involved in glutamate signaling. A second cohort of psychiatric samples was also tested by quantitative PCR using the primer/probe sets for EFNA5, GLUR7, CACNG2 and AKAP5, and samples with aberrant copy number were found at three of the four loci (GLUR7, CACNG2 and AKAP5). Further scrutiny of these regions may reveal insights into the etiology and genetic risk factors for these complex psychiatric disorders.

A Kinase Anchor Proteins↗

PubMed1998

Individual variation in neuron number predicts differences in the propensity for avian vocal imitation.

Avian song learning involves memorizing and reproducing song material produced by conspecifics. In several species song repertoire size correlates with the overall volume of two song-related brain regions, the HVc (acronym used as the proper name) and the robust nucleus of the archistriatum (RA). We raised male zebra finches with two adult tutors and found that individual differences in HVc volume and neuron number correlated positively with differences in the number of tutor syllables accurately copied. These results were replicated in a second study. The relationship between RA volume and song learning was similar, but less robust. Importantly, total repertoire size (number of song syllables) did not correlate significantly with anatomical measures of either the HVc or RA. Because previous work suggests that the volume and neuron number of these regions are not regulated by song learning, it is possible that naturally occurring variation in neuron number constrains how much song material can be copied or reproduced.

Animals↗

PubMed2003

Genesis of variants of Vibrio cholerae O1 biotype El Tor: role of the CTXphi array and its position in the genome.

The gene encoding cholera toxin, the principal virulence factor of Vibrio cholerae, is encoded by a filamentous, lysogenic bacteriophage known as CTXphi. The genome of V. cholerae, the host for CTXphi, consists of two chromosomes, one large and one small. Here, it is shown that localization and array of CTX prophage DNA in either the large or small chromosome of V. cholerae is likely to be one of the reasons for the emergence of O1 biotype El Tor variants isolated just before and after the V. cholerae O139 cholera outbreak in 1992. Analyses of the organization of the CTX region of the genome of pre-O139 El Tor strains revealed that these strains carry two distinct CTX prophages integrated in the small chromosome in tandem: CTX(ET), the prophage having a conserved NotI site in its repeat sequence segment which seems to be specific for the El Tor strains so far examined, followed by CTX(calc)-like genome, the prophage found in recent O139 clinical isolates from Calcutta. In sharp contrast, in post-O139 El Tor strains only one copy of the CTX(ET) prophage was found to be integrated in the large chromosome. To the authors' knowledge, the presence of CTX prophage in the small chromosome of O1 El Tor strains has not been reported previously. It is also shown that the difference in the CTX copy number and the position of the bacteriophage on the genomes of pre- and post-O139 El Tor strains have an effect on cholera toxin production. While a pre-O139 strain produced maximum cholera toxin in yeast extract/peptone medium at 30 degrees C, a post-O139 El Tor strain showed maximal yield at 37 degrees C, indicating differential regulation of cholera toxin between the strains. It appears from this study that the variation in the integration site of the CTX prophage, its copy number and the presence of diverse phage genomes in V. cholerae O1 biotype El Tor may be strategically important for generating variants with subtle phenotypic modulations of virulence factor production in this longest-ruling seventh pandemic strain.

Bacteriophages↗

PubMed1995

The MAR-Mediated Reduction in Position Effect Can Be Uncoupled from Copy Number-Dependent Expression in Transgenic Plants.

To study the role of matrix-associated regions (MARs) in establishing independent chromatin domains in plants, two transgenes were cloned between chicken lysozyme A elements. These transgenes were the neomycin phosphotransferase (NPTII) gene under control of the nopaline synthase (nos) promoter and the [beta]-glucuronidase (GUS) gene controlled by the double cauliflower mosaic virus (dCaMV) 35S promoter. The A elements are supposed to establish an artificial chromatin domain upon integration into the plant DNA, resulting in an independent unit of transcriptional regulation. Such a domain is thought to be characterized by a correlated and position-independent, hence copy number-dependent, expression of the genes within the domain. The presence of MARs resulted in a higher relative transformation efficiency, demonstrating MAR influence on NPTII gene expression. However, variation in NPTII gene expression was not significantly reduced. The selection bias for NPTII gene expression during transformation could not fully account for the lack of reduction in variation of NPTII gene expression. Topological interactions between the promoter and A element may interfere with the A element as a domain boundary. In contrast, the GUS gene on the same putative chromatin domain showed a highly significant reduction in variation of gene expression, as expected from previous results. Surprisingly, no copy number-dependent GUS gene expression was observed: all plants showed approximately the same GUS activity. We concluded, therefore, that dCaMV 35S-GUS gene expression in mature tobacco plants is regulated by some form of dosage compensation.

Journal Article↗

PubMed2005

High-resolution gene copy number and expression profiling of human chromosome 22 in ovarian carcinomas.

Previous low-resolution studies of chromosome 22 in ovarian carcinoma have suggested its involvement in the development of the disease. We report a high-resolution analysis of DNA copy number and gene expression of 22q in 18 ovarian carcinomas using a 22q-specific genomic microarray. We identified aberrations in 67% of the studied tumors, which displayed 3 distinct gene copy number profiles. The majority of the cases (11 of 18) demonstrated heterozygous terminal deletions of various sizes, the smallest of which was 3.5 Mb. The second profile, detected in 3 tumors, revealed the coexistence of heterozygous deletions and different patterns of low-copy-number gain that involved the proximal half of 22q. The latter finding has not been reported previously in ovarian carcinoma. One case displayed a continuous deletion encompassing the entire 22q, consistent with monosomy 22. Furthermore, we compared the results with the available data on these tumors by using cDNA microarrays to define the degree of correlation between abnormalities at the DNA level and variation in mRNA expression. By a comparison with the expression data, we were able to identify 21 deleted genes showing low mRNA levels and 12 amplified genes displaying elevated gene expression, several of which play roles in cell cycle control and the induction of apoptosis. Our results indicated significant correlation between DNA copy number aberrations and variation in mRNA expression. We also identified several regions and candidate genes on 22q that should be studied further to determine their role in the development of ovarian cancer.

Adenocarcinoma, Clear Cell↗