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PubMed2003

Diversity of group II introns in the genome of Sinorhizobium meliloti strain 1021: splicing and mobility of RmInt1.

The number and diversity of known group II introns in eubacteria are continually increasing with the addition of new data from sequencing projects, but the significance of these introns in the evolution of bacterial genomes is unknown. We analyzed the main features of the group II introns present in the genome of the soil microorganism Sinorhizobium meliloti (strain 1021), the nitrogen-fixing symbiont of alfalfa, the DNA sequence of which was recently determined. Strain 1021 harbors three different classes of group II introns: RmInt1, of bacterial class D; SMb2147/SMb21167, which cluster within bacterial class C; and SMa1875, the phylogenetic class of which is uncertain. The group II introns SMb2147/SMb21167 and SMa1875 are widely distributed in S. meliloti, but are present in lower copy numbers than RmInt1. Strain 1021 harbors three copies of RmInt1, which is pSym-specific. Although RmInt1 is spliced in strain 1021, mobility assays suggested that, in contrast to other S. meliloti strains, the genetic background of strain 1021 does not support intron homing events.

Bacterial Proteins↗

PubMed1996

Sequence divergence in a family of variant surface glycoprotein genes from trypanosomes: coding region hypervariability and downstream recombinogenic repeats.

The surface of the parasitic protozoan Trypanosoma brucei spp. is covered with a dense coat consisting of a single type of glycoprotein molecule, the variant surface glycoprotein (VSG). There may be as many as 1,000 genes for VSG within the genome of T. brucei, and the switch of expression from one to another is the phenomenon of antigenic variation. As an approach to understanding the evolution of VSG genes we have determined the genomic DNA sequences of the eight genes encoding the variant surface glycoprotein 117 (VSG) family. From these data we have observed a number of features concerning the relationships between these genes: (1) there is a region of high variability confined to the N-terminus of the coding sequence, and comparison of the sequences with the available X-ray diffraction crystal structures suggests that two of the most variable stretches within the N-terminal domain are present on surface-exposed loops, indicating a role for epitope selection in evolution of these genes; (2) the 29 nucleotides surrounding the splice acceptor site are absolutely conserved in all eight 117 VSG genes; (3) numerous insertion/deletion mutations are located within or immediately downstream of the C-terminal protein-coding sequences: (4) within 500 bp downstream of the insertion/deletion mutations are one or two copies of a repeat motif highly homologous to the recombinogenic 76-bp repeat sequences present upstream of many VSG basic copy genes and the expression-linked copy.

Amino Acid Sequence↗

PubMed2003

Copy number of P elements, KP/full-sized P element ratio and their relationships with environmental factors in Brazilian Drosophila melanogaster populations.

The P transposable element copy numbers and the KP/full-sized P element ratios were determined in eight Brazilian strains of Drosophila melanogaster. Strains from tropical regions showed lower overall P element copy numbers than did strains from temperate regions. Variable numbers of full-sized and defective elements were detected, but the full-sized P and KP elements were the predominant classes of elements in all strains. The full-sized P and KP element ratios were calculated and compared with latitude. The northernmost and southernmost Brazilian strains showed fewer full-sized elements than KP elements per genome, and the strains from less extreme latitudes had many more full-sized P than KP elements. However, no clinal variation was observed. Strains from different localities, previously classified as having P cytotype, displayed a higher or a lower proportion of KP elements than of full-sized P elements, as well as an equal number of the two element types, showing that the same phenotype may be produced by different underlying genomic components of the P-M system.

Animals↗

PubMed1996

The clp (CS31A) operon is negatively controlled by Lrp, ClpB, and L-alanine at the transcriptional level.

Biosynthesis of the Escherichia coli CS31A surface antigen was subject to phase-variation control and repressed by L-alanine. Nucleotide sequence analysis of the clp operon, encoding the biosynthesis of CS31A, revealed the presence of a regulatory gene, clpB. The amino acid sequence of the regulatory protein ClpB showed similarity to the primary structure of PapB, FaeB and AfaA, involved in the regulation of expression of Pap, K88, and Afa-3 fimbriae, respectively. The clp regulatory region contained two deoxyadenosine methylase sites (GATC-I and GATC-II). The leucine-responsive regulatory protein (Lrp) was required for specific methylation inhibition of the GATC-II site. The activity of the clp promoter was monitored in a clp-lacZYA single-copy fusion. The cloned DNA used in this study did not contain a related papl gene. In these conditions, we showed, as expected, that phase variation did not occur. However, transcription of the clp operon was negatively controlled by ClpB and Lrp, and was maximal in the absence of Dam methylase. In the presence of AfaF, a Papl equivalent, the phase-variation control was restored. We concluded that two regulatory mechanisms were superimposed to control the clp expression. Phase variation, mediated by Lrp and a Papl equivalent, controlled the number of cells producing CS31A in a single colony. The second mechanism, described in this report, was mediated by ClpB and Lrp and controls the level of CS31A produced by a single cell. Furthermore, we showed that L-alanine reduced, by about twofold, the clp promoter activity independently of a Papl equivalent, ClpB, Lrp or Dam methylase. In addition, the presence of L-alanine prevented the phase-variation control mediated by AfaF.

Alanine↗

PubMed1994

Rodent BC1 RNA gene as a master gene for ID element amplification.

ID elements are short interspersed repetitive DNA elements (SINEs) which have amplified in rodent genomes via retroposition, a process involving an RNA intermediate. BC1, an abundant ID-related transcript, is transcribed from a conserved, single-copy gene in rodents. The gene encoding BC1 RNA represents one of the earliest and possibly the first ID-containing sequence. Comparison of consensus sequences of each rodent ID with its corresponding BC1 RNA gene showed that the variations of BC1 RNA within rodents corresponded to specific changes within the ID consensus sequence for each rodent species. This supports the hypothesis that the BC1 gene is a master gene responsible for the amplification and evolution of ID elements. The rat ID family consists of at least four subfamilies, with the oldest subfamily having been derived from the BC1 RNA. The other three subfamilies appear to have been derived from a new master gene(s), which has been responsible for the large increase in ID element copy number within the rat genome. We have found that the guinea pig genome contains two copies of the BC1 gene, apparently the result of a DNA-mediated duplication event. Both of these guinea pig BC1 genes have a conserved TATA-like element in the 5' flanking region and have contributed to guinea pig ID amplifications.

Animals↗

PubMed1993

Mitotic stability and meiotic variability of the (CAG)n repeat in the Huntington disease gene.

The gene causing Huntington's disease, an autosomal dominantly inherited, neurodegenerative disorder, has been identified recently. The corresponding mutation is involving an expansion in the number of (CAG)n repeats in the coding region of the Huntington's disease gene on chromosome 4. In this report, we demonstrate the length variation of the repeat in 513 non-HD chromosomes from normal individuals and HD patients showing 23 alleles with 11 to 33 repeats. Analyzing the inheritance of the (CAG)n stretch we found meiotic instability for HD alleles ([CAG]40 to [CAG]75) with a mutation frequency of approximately 0.7, while in 431 meioses of normal alleles only two expansions were identified. The risk of expansion during spermatogenesis is enhanced compared to oogenesis explaining juvenile onset by transmission from affected fathers. Further, the number of (CAG)n copies in an affected individual in relation to the sex of the transmitting parent was evaluated and no significant differences were found. No mosaicism or differences in the repeat lengths were observed in the DNA from different tissues including brain and lymphocytes of two HD patients indicating mitotic stability of the mutation. Therefore, the determination of the repeat number in the DNA of blood lymphocytes is probably representative of all tissues in a patient.

Alleles↗

PubMed2000

Fluorescence in situ hybridization (FISH) for detection of HER-2/neu amplification in breast cancer: a multicenter portability study.

Amplification and/or overexpression of HER-2/neu has been shown to be both a prognostic and predictive marker in breast cancer. Recent studies have also confirmed the efficacy of Herceptin (trastuzumab) as adjuvant therapy for patients with overexpression of HER-2/neu. Therefore, it is critical that precise and reproducible assays be used in the clinical laboratory setting for determination of the HER-2/neu status in patients with breast cancer. The objective of this study was to determine the portability (reproducibility between different institutions) of the PathVysion HER-2 fluorescence in situ hybridization (FISH) assay used for detection of amplification of the HER-2/neu gene in formalin-fixed, paraffin-embedded tissue sections of invasive ductal carcinoma of the breast. Study specimens consisted of one breast tumor with a normal HER-2/neu copy number, two tumors with a low level, and one tumor with a high level of HER-2/neu amplification. The PathVysion HER-2 assay was shown to be highly reproducible on different assay days (n = 3) and between different institutions (n = 5) in the detection of amplification of the HER-2/neu gene in routinely processed clinical specimens of breast carcinoma. In addition, this study examined the feasibility of enumerating FISH signals in 20 nuclei in contrast to 60 nuclei per specimen. Although a modest increase in variation was observed when analyzing 20 compared to 60 nuclei, the mean ratios were similar. Therefore, analysis of as few as 20 nuclei with this FISH HER-2/neu assay may be sufficient for determining the amplification level of the HER-2/neu gene.

Antimetabolites, Antineoplastic↗

PubMed2001

Etiology of differences in hematocrit between males and females: sequence-based polymorphisms in erythropoietin and its receptor.

INTRODUCTION: Males have a higher hematocrit (Hct) than females. The cause of this gender-based difference is unclear. We sought to determine whether polymorphisms of the erythropoietin (EPO) gene or of its receptor (EPOR) explain this situation. METHODS: We designed primers for the EPO and EPOR genes. Previously undescribed polymorphisms were found based on band migration on polyacrylamide gel, and when then sequenced. The distribution of these polymorphisms was studied in a population of 819 non-iron-deficient, healthy blood donors. To test the gender differences, analysis was done based on groups defined by Hct levels. The chi-square statistic was used to compare the frequency differences between groups, with P < .05 considered statistically significant. RESULTS: We found previously reported polymorphisms in both the EPO and EPOR genes. Sequence analysis showed that the EPO polymorphism was due to a difference in the repeat copy number of the tetranucleotide cytosine adenine cytosine thymine (CACT) at position 2153. A previously undescribed 12th allele was found for the EPOR polymorphic site. Statistical analysis showed that the EPOR alleles, EPORA1 and EPORA10, were present at a significantly higher frequency in females than in males (P = .027 and P = .041, respectively), and EPOR5 was found less frequently in females than in males (P = .048). The allelic frequency of the EPO polymorphism was not significantly different by gender or Hct groups. DISCUSSION: These results suggest that the variation of Hct level by gender may have a genetic basis. The sequence-based polymorphism for EPOR may be partly responsible for this gender-based variation in Hct level. These findings offer new clues to understanding Hct variation in the general population and to elucidating mechanisms of controlling Hct levels.

Adult↗

PubMed1994

The relationship between DNA structural variation and activities of P elements in P and Q strains of Drosophila melanogaster.

To characterize the relationship between P element activities and their structures, we cloned P elements from genomic libraries of three isogenic P and Q strains derived from natural populations in Japan. These P elements were mapped with BamHI, AvaII and PstI and were classified by their size. The majority of P elements cloned were classified as either complete or relatively small P elements rather than medium size. The numbers of full length (2.9 kb) P elements per haploid genome of NP280 (P), AK194 (weak P) and WY113 (Q) were at least four, five and one, respectively. However, the 2.9 kb P element of WY113 was thought to be defective since this strain has no transposase activity. In our previous work, we demonstrated that the ORF 3-deleted P element is essential for P cytotype determination in WY113. A similar P element also exists in NP280, and this may have an important role for P cytotype determination in this strain. Two and one copies of the KP element, a deletion derivative of the P element, were found in NP280 and AK194, respectively. One of four complete P elements in NP280 was fully sequenced, and the base sequence was completely identical to that of p pi 25.1 originally derived from the U.S.A. This result is consistent with the notion that these P elements have a relatively recent origin in Drosophila melanogaster.

Animals↗

PubMed1991

Heterologous gene expression in continuous cultures of budding yeast.

In a previous paper we have studied the expression of beta-galactosidase from Escherichia coli, driven from the inducible GAL1-10/CYC1 hybrid promoter, in batch cultures of budding Saccharomyces cerevisiae and have described operating conditions for maximal productivity. In this paper we show that the plasmid instability in continuous cultures can be overcome by utilizing appropriate selection markers and a high copy number vector. The maximal level of expression is influenced by the dilution rate. Moreover, enzyme accumulation appears to depend also upon the degree of oxygenation. A possible explanation of these modulations is discussed, taking into account the interactions of the UAS-GAL and TATA-CYC1 elements.

Escherichia coli↗

PubMedDate not supplied

Association of short alleles of a VNTR of the serotonin transporter gene with anxiety symptoms in patients presenting after deliberate self harm.

The polymorphism of a variable number tandem repeat (VNTR) region of the serotonin transporter gene consists of three alleles containing, respectively, 9 (STin2.9), 10 (STin2.10) and 12 (STin2.12) copies of a repetitive element. The frequencies of the three alleles in 384 individuals presenting after deliberate self harm were the same as a group of 346 controls. However, ratings of anxiety (and state anger) were higher in those patients with genotypes including the shorter repetitive elements. The findings support the hypothesis that, in this group of patients with low rates of severe psychiatric disorder, allelic variation in the serotonin transporter gene may contribute to the expression of anxiety symptoms.

Aggression↗

PubMed1999

An international collaborative study on the detection of an HIV-1 genotype B field isolate by nucleic acid amplification techniques.

An international collaborative study to assess inter-laboratory variation in the sensitivity of gene amplification assays for the detection of HIV-1 RNA sequences was conducted using a panel of eight duplicate dilutions of an HIV-1 genotype B clinical isolate and negative control samples. Twenty-five laboratories participated in the study and used a variety of in-house assays and commercial assay systems. With few exceptions, the assays were more sensitive than a p24 antigen assay. Overall, the PCR-based Amplicor Monitor assay was the most sensitive and gave the highest mean copy number for any one sample. Some of the in-house assays gave results comparable with the Monitor assay whilst the NASBA and bDNA assays appeared to be less sensitive. As a result of this study, an HIV-1 Working Reagent for the standardisation of nucleic acid amplification assays was developed and assessed in a subsequent study. Similar differences in sensitivity between the different assay systems was observed. The discrepancies in viral copy number obtained using the Working Reagent highlights the need for an International Standard against which all Working Reagents may be calibrated.

Adult↗

PubMed1992

Cyclic GMP-activated channels of salamander retinal rods: spatial distribution and variation of responsiveness.

1. Patch-clamp methods were used to investigate the areal density and spatial location of cyclic GMP-activated channels in the surface membrane of salamander rod outer segments. 2. The density of active channels (i.e. channels able to respond to cyclic GMP) in patches excised from outer segments was determined from the number of active channels, N, and the membrane area, A. N was estimated from the current induced by a saturating concentration of cyclic GMP, while A was estimated from the electrical capacitance of the patch. 3. In patches excised from forty-one isolated outer segments prepared in the light the active channel density varied over a remarkable range: 0.34-629 microns-2, with a mean of 166 microns-2. Density was not correlated with patch area in this or any of the conditions studied. 4. The spatial distribution of open channels on the outer segment of a transducing rod was measured by recording the local dark current at various positions with a loose-patch electrode. The apparent density of open channels varied by only about +/- 50% around the circumference of the outer segment and up and down its length. This indicates that the wide range of densities in excised patches did not result from sampling a non-uniform spatial distribution of channels. 5. Patches excised from sixteen dark-adapted whole cells with healthy appearances and saturating light responses of normal size had active channel densities of 1.1-200 microns-2, with a mean of 60 microns-2. Patches from twenty light-adapted whole cells had similar densities. Many densities from the whole cells were much lower than expected. This, and the wide variation in densities, suggests that obtaining a patch often lowered the density of active channels. The number of channels in a patch was quite stable from 1 s to 30 min after excision, ruling out progressive denaturation or adsorption of channels to the glass as a cause for this effect. 6. The mean active channel density in patches excised from whole cells was lower with calcium present in the external solution than with calcium absent (80 vs. 152 microns-2, n = 36 and 30 respectively). 7. We conclude that copies of the channel protein were present at a density of at least 650 microns-2 in the surface membrane of the outer segment and that the distribution of channels was fairly uniform on a 1 micron scale.(ABSTRACT TRUNCATED AT 400 WORDS)

Adaptation, Ocular↗

PubMed1987

Genetic variations in kinetic constants that describe beta-glucuronidase mRNA induction in androgen-treated mice.

The kinetics of beta-glucuronidase mRNA induction by androgen in mouse kidney were determined for A, B, and CS haplotypes of the beta-glucuronidase gene. After a lag period, the kinetics of mRNA (R) induction are approximated by the turnover equation dR/dt = k1 - k2R. The A haplotype differs from the B primarily in the duration of the lag period and in k1, the rate constant determining the initial slope of the induction curve. The CS haplotype differs from B primarily in k2, the first-order rate constant that determines the half-time for induction. None of the haplotypes differs significantly in the half-life of beta-glucuronidase mRNA as measured by deinduction. Thus, there was no correlation between the half-time or extent of induction and the half-life of the RNA. Comparing half-times for induction with the half-life of the mRNA suggests that message stabilization can at most account for only part of the induction. We conclude that transcriptional activation of the beta-glucuronidase gene must be an important component of induction. Estimating absolute numbers of mRNA molecules and absolute rates of gene transcription, it appears that before induction there is approximately one molecule of beta-glucuronidase mRNA per cell and that each gene copy is transcribed once every 35 to 40 h. Depending on the haplotype examined, after induction, mRNA goes up to 80 to 400 molecules per induced cell. In the A haplotype, which has the highest induction, this corresponds to one transcript from each gene every 6 min if there is no induced stabilization of beta-glucuronidase mRNA, and one every 30 min if there is. Thus, it seems unlikely that more than one transcript is ever being synthesized at the same time from the beta-glucuronidase gene.

Animals↗

PubMed1985

An evolutionary comparison of homologous mitochondrial plasmid DNAs from three Neurospora species.

We have discovered a mitochondrial DNA plasmid in N. crassa 516 (Roanoke, LA) which is homologous to those previously described from N. intermedia 435 (Fiji) and N. tetrasperma 2510 (Hanalei, HA). Subsequent analysis by DNA-DNA hybridization showed that 6 of 14 other Louisiana N. crassa isolates possessed plasmids homologous to these three plasmids, but at lower copy number. Plasmids from the three named strains were studied to examine possible plasmid diversity within each isolate, the extent of the homology between the plasmids, and the possibility that these plasmids could be inherited separately from their host mitochondria. Comparison of cloned plasmids and covalently closed circular mitochondrial DNA showed that only one plasmid line was present in each of the three intensively studied isolates. DNA-DNA hybridization and restriction endonuclease site mapping showed that the mitochondrial plasmids from the three species were very similar; most of the variation was due to presumed nucleotide substitutions. Plasmids judged identical by our analysis were found in different species. The distribution of the homologous plasmids in nature and the presence of these identical plasmids in different species, suggested that these plasmids could be transmitted between isolates independently of their host mitochondria.

Biological Evolution↗

PubMed1985

Transposable element-induced response to artificial selection in Drosophila melanogaster.

The P family of transposable elements in Drosophila melanogaster transpose with exceptionally high frequency when males from P strains carrying multiple copies of these elements are crossed to females from M strains that lack P elements, but with substantially lower frequency in the reciprocal cross. Transposition is associated with enhanced mutation rates, caused by insertion and deletion of P elements, and chromosome rearrangements. If P element mutagenesis creates additional variation for quantitative traits, accelerated response to artificial selection of progeny of M female female X P male male strain crosses is expected, compared with that from progeny of P female female X M male male strain crosses.--Divergent artificial selection for number of bristles on the last abdominal tergite was carried out for 16 generations among the progeny of P-strain males (Harwich) and M-strain females (Canton-S) and also of M-strain males (Canton-S) and P-strain females (Harwich). Each cross was replicated four times. Average realized heritability of abdominal bristle score for the crosses in which P transposition was expected was 0.244 +/- 0.017, 1.5 times greater than average heritability estimated from crosses in which transposition was expected to be rare (0.163 +/- 0.010). Phenotypic variance of abdominal bristle score increased by a factor of four in lines selected from M female female X P male male crosses when compared with those selected from P female female X M male male hybrids. Not all quantitative genetic variation induced by P elements is additive. A substantial fraction of nonadditive genetic variation is implicated by chromosomal analysis, which demonstrates deleterious fitness effects of the mutations when homozygous.--Several putative "quantitative" mutations were identified from chromosomes extracted from the selected lines; these will form the basis for further investigation at the molecular level of the genes controlling quantitative inheritance.

Animals↗

PubMed1999

Abnormal chromosome 8 copy number in stage I to stage IV breast cancer studied by fluorescence in situ hybridization.

To test the hypothesis that the frequency of abnormal chromosome 8 copy number increases with the severity of the disease as defined by an increase in clinical stage, we conducted a fluorescence in situ hybridization (FISH) study of a sample of 42 breast cancer specimens utilizing a protocol that was optimized by our laboratory. Cytogenetic results, obtained from blinded analyses of archival specimens, demonstrated that the higher clinical stages (i.e., stages III and IV) yield higher frequencies of abnormal chromosome 8 copy number. Specifically, 45.45% and 50% of the stage I and stage II cases, respectively, were abnormal, whereas 63.64% and 60% of the stage III and stage IV cases, respectively, were abnormal for chromosome 8 copy number. The overall frequency of abnormal chromosome 8 copy number was 54.76% (23 of 42 tumors studied). When the results of a control probe were taken into account, 34.78% (8 of 23) of the abnormal cases were trisomic, whereas the remaining cases were likely triploid. Thus, the present data not only established that chromosome 8 trisomy is a recurrent finding in breast cancer, but also confirmed a higher frequency of occurrence of abnormal chromosome 8 copy number with the higher clinical stages. Future experiments utilizing additional specimens in this laboratory and from other laboratories are necessary to confirm and extend the findings of the present study.

Aneuploidy↗

PubMed1994

Evidence that most human Alu sequences were inserted in a process that ceased about 30 million years ago.

The primate Alu interspersed repeats can be subdivided into classes on the basis of shared nucleotides at a set of diagnostic positions. Each of the classes of Alu sequences is apparently the result of past retrotransposition of transcripts of highly conserved class-specific source genes that differed from each other at the diagnostic positions. The nucleotides at the majority of positions are identical among the source genes and therefore were identical among all of the Alu sequences at the time of their insertion. These CONSBI (conserved before insertion) positions are useful because the changes that have occurred after insertion are recognizable and the divergence resulting from nucleotide substitutions, insertions, and deletions is informative. The divergence of Alu sequences at the CONSBI positions is a measure of the time since a class was inserted. The greatest majority of Alu sequences are in one class (identified as class II), and it is particularly suitable for such examination, since nearly full-length sequences are now known for nearly a thousand members of this class. The average divergence of class II members indicates that the class has an average age of about 40 million years. The distribution in divergence of class II accurately fits a sum of two Poisson distributions. The implication is that class II Alu sequences were derived from two massive past events of insertion of many Alu sequences. In this model the younger subset of class II sequences (corresponding to about 300,000 copies in the genome) has an average divergence of 5% at CONSBI positions. The older set of class II sequences (corresponding to about 150,000 genomic copies) has a 9% average divergence. Based on the drift rate of primate DNA sequences, the events of insertion probably occurred 30-50 million years ago. The goodness of the fit to the Poisson distribution indicates that no significant number of members of class II have been inserted since 30 million years ago.

Animals↗