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PubMed1985

Comparison of MHC genes among distantly related members of the genus Mus.

The genomic content of class I and class II MHC DNA sequences in a variety of wild mice has been analyzed. The panel of mice includes members of three subgenera of the genus Mus. By genomic hybridization with the use of a variety of DNA probes, both class I and class II DNA sequences appear to be conserved in all of the species examined. However, the number of class I DNA sequences differs among the species. Furthermore, this variation appears to result from differential increases within subsets of class I genes. These data suggest that the class I multigene family is dynamic and changing over short periods of evolutionary time. In contrast, none of the class II genes appears to vary in copy number. More extensive polymorphism was noted amongst the class II beta genes than the alpha genes. Interestingly, the genomic sequence corresponding to E beta 2 is highly conserved, leading to the prediction that it is a genetically functional sequence.

Animals↗
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↗
PubMed1995

Segment selection during 'phase variation'-type regulation of gene replacement mediated by FLP recombinase in the yeast Saccharomyces cerevisiae.

FLP recombinase has recently been used as a tool to direct the exchange between invertible DNA segments, called 'Phase variation'-type regulation of gene replacement in eukaryotic cells. Using an appropriate selective medium, positive segment selection was shown to be efficient during the regulation of gene replacement. The efficiency was determined from the copy number ratio of invertible segments with the use of the neomycinphosphotransferase II (NPTII) gene bearing invertible segments located on the episomal yeast plasmid, and the resident 2-microns circle. Without the selection the segments copy number ratio was retained in growing cells. The results obtained are an evidence for the efficiency of positive segment selection during the 'Phase variation'-type regulation of gene replacement in eukaryotic cells.

DNA Nucleotidyltransferases↗
PubMed1994

Quantitative analysis of gene expression in different tissues by template-calibrated RT-PCR and laser-induced fluorescence.

RT-PCR is widely used to study gene transcription in many biological systems. Despite the development of a variety of, at times complex, procedures, quantitation of RT-PCR remains difficult, particularly when comparing RNA from different tissues or very small samples. In the procedure described here, we calibrate input cDNA through incorporation of trace label. PCR product is generated from equal amounts of cDNA with fluoresceinated primers, size fractionated, and quantitated by laser-induced fluorescence in an automated DNA sequencer. Eliminating variation in input cDNA resulted in reliable noncompetitive PCR quantitation from templates equivalent to > or = 50 pg of total RNA. Using the example of beta-glucuronidase, a low-copy-number housekeeping gene, we have drawn a map of differential gene expression for this protein in various rat tissues.

Animals↗
PubMed1986

The simple repeat poly(dT-dG).poly(dC-dA) common to eukaryotes is absent from eubacteria and archaebacteria and rare in protozoans.

Genomic DNA from a wide variety of prokaryotic and eukaryotic organisms has been assayed for the simple repeat sequence poly(dT-dG).poly(dC-dA) by Southern blotting and DNA slot blot hybridizations. Consistent with findings of others, we have found the simple alternating sequence to be present in multiple copies in all organisms in the animal kingdom (e.g., mammals, reptiles, amphibians, fish, crustaceans, insects, jellyfish, nematodes). The TG element was also found in lower eukaryotes (Saccharomyces cerevisiae, Neurospora crassa, and Dictyostelium discoideum) and at a much lower frequency in protozoans (Oxytricha fallux and Tetrahymena thermophila). The sequence was also repeated in high copy number in a higher plant (Zea mays) as well as at very high levels in a unicellular green alga (Chlamydomonas reinhardi). Although the copy number of the repeat per haploid genome was generally proportional to genome size, there was a greater-than-1,000-fold variation in the number of (TG)25/100-kb genomic DNA. By contrast, no eu-or archaebacterium--including Myxococcus xanthus, whose life cycle is very similar to that of the slime mold Dictyostelium discoideum, and Halobacter volcanii, whose genome contains other repeated sequences--was found whose genomic DNA contained this sequence in detectable amounts. A computer search also failed to find the TG element in human mitochondrial DNA.

Animals↗
PubMed1992

Heteroplasmy of short tandem repeats in mitochondrial DNA of Atlantic cod, Gadus morhua.

The mitochondrial DNA of the Atlantic cod (Gadus morhua) contains a tandem array of 40-bp repeats in the D-loop region of the molecule. Variation among molecules in the copy number of these repeats results in mtDNA length variation and heteroplasmy (the presence of more than one form of mtDNA in an individual). In a sample of fish collected from different localities around Iceland and off George's Bank, each individual was heteroplasmic for two or more mtDNAs ranging in repeat copy number from two (common) to six (rare). An earlier report on mtDNA heteroplasmy in sturgeon (Acipenser transmontanus) presented a competitive displacement model for length mutations in mtDNAs containing tandem arrays and the cod data deviate from this model. Depending on the nature of putative secondary structures and the location of D-loop strand termination, additional mechanisms of length mutation may be needed to explain the range of mtDNA length variants maintained in these populations. The balance between genetic drift and mutation in maintaining this length polymorphism is estimated through a hierarchical analysis of diversity of mtDNA length variation in the Iceland samples. Eighty percent of the diversity lies within individuals, 8% among individuals and 12% among localities. An estimate of theta = 2N(eo) mu greater than 1 indicates that this system is characterized by a high mutation rate and is governed primarily by deterministic dynamics. The sequences of repeat arrays from fish collected in Norway, Iceland and George's Bank show no nucleotide variation suggesting that there is very little substructuring to the North Atlantic cod population.

Animals↗
PubMed1989

A tandem repeat sequence found in a heterogeneous fragment of UL of herpes simplex virus type 1.

We found a tandem repeat sequence in the region (designated BS7) in which restriction fragments BamHI D and SalI B overlap each other, near the centre of the unique long sequence (UL) of the herpes simplex virus type 1 (HSV-1) strain F genome. The SmaI physical map of BS7 was constructed, and the position of a heterogeneous SmaI subfragment from HSV-1 isolates and plaque-purified clones from a single strain was defined on the map. The maximum size difference in the SmaI subfragment was estimated to be 600 bp between these isolates and 100 bp between the clones. The 0.23 kb SmaI subfragment recloned from BS7 was sequenced, and was shown to contain a tandem repeat sequence consisting of 15 units of 12 bp, 5' TTGGGGCTGGGG 3'. These results suggest that the fragment length heterogeneity in the UL of HSV-1 isolates and clones is attributable to copy number variation of the tandem repeat sequence.

Base Sequence↗
PubMed1994

Sequence variation in the capsid protein genes of human papillomavirus type 16.

We have cloned and sequenced the L1 and L2 genes from human papillomavirus type 16 (HPV16) DNA-containing cervical cytology samples collected from the U.K. and Trinidad. Samples containing high copy numbers of HPV16 DNA were selected as being likely to contain fully functional virus DNA molecules in an episomal state, rather than in an integrated and possibly altered state. In comparison with the previously published sequence of HPV16 isolated from an invasive cancer a variety of differences were detected in both L1 and L2. The pattern of changes appears to be different in samples from the two geographic regions. One of the differences (resulting in D at position 202 of the L1 protein) reported recently to be functionally important for virus particle assembly was found to occur in all the samples examined. Variations in L1 found within known immunoreactive regions or hydrophobic domains should be taken into account in design of prophylactic vaccines for HPV16 based on virus-like particles. All variations within L2 protein were found in hydrophilic domains in the carboxy-terminal half of L2. These positions were highly variable among other types of papillomavirus and are located outside the known L2 immunoreactive region.

Amino Acids↗
PubMed1995

Construction of RNA standards for high-resolution automatic product analysis in quantitative competitive RT-PCR.

The exponential character of PCR amplification may compromise quantitative assays because it multiplies minor sample-to-sample variations. To overcome these problems, several authors have used recombinant standard DNA or RNA molecules to be spiked into the samples in a dilution series of known copy numbers before co-amplification by PCR. To obtain an equal efficacy of reverse transcription and PCR amplification, standard and template molecules should be highly homologous. However, the limited resolution of commonly used agarose gel electrophoresis requires rather large differences in size and nucleotide sequence to separate both molecules from each other after PCR. Due to a much higher resolution, automatic post-PCR analyzing systems based on laser-induced fluorescence may help to overcome these difficulties. For using the capabilities of these systems in quantitative competitive RT-PCR, we developed a protocol to construct recombinant RNA standard molecules that only differ from the target sequence by a small deletion of 8 nucleotides. It is based on PCR-induced mutagenesis and solid-phase in vitro transcription. This protocol was applied to quantify multidrug resistance gene (MDRI) mRNA in malignant cells, but it can easily be adapted to any gene of interest.

Base Sequence↗
PubMed1986

Evaluating quantitative variation in the genome of Zea mays.

Genomic diversity within the species Zea mays has been examined by measuring the variation in the repetitive component of the nuclear genome among North American inbred lines and varieties. This was done by preparing a set of clones of repetitive maize sequences that differ in function, molecular arrangement and multiplicity and then using these as probes for quantitative hybridization to DNA from various maize genotypes. The comparison showed that the majority of repeated sequences are markedly variable in copy number among the ten maize strains tested. The clone sample contained the rDNA and 5S genes, the major repeat of the chromosome knobs, sequences functioning as origins of DNA replication in yeast (ARS sequences) and randomly cloned sequences of unknown function and chromosomal location. The sequences ranged in reiteration frequency from 200 to greater than 10(5) copies and included both tandemly arrayed and dispersed repeats. The copy numbers were measured by hybridizing labeled cloned sequences to aliquots of high molecular weight genomic DNA that were applied to nitrocellulose filters through a slotted template (slot blotting). The hybridization signal on an autoradiogram occurred in a narrow band that could be scored reliably with a densitometer. This provided a rapid method of determining the abundance of particular repeated sequences in individual plants and plant populations. Using this technique, we found that the copy number of repeated sequences of all types generally varied among the strains by two- to threefold, although at least one sequence showed no detectable variation. In contrast to the variability found between strains, individuals within an inbred line or variety were found to be indistinguishable in terms of specific sequence multiplicity. Each genotype has a different pattern of copy numbers for the set of repeated sequence clones, and this pattern is characteristic of all individuals of a particular genotype. The data also show that the copy number of each sequence varies independently. No strains had uniformly high or low copy numbers for the entire set of probes.

DNA↗
PubMed1986

Variation in the number of alpha-globin loci in sheep.

Southern blot analysis was used to compare sheep and goat restriction-endonuclease maps of the DNA region containing the alpha-globin genes. The identical digestion patterns observed in both species with three endonucleases (BamHI, BstEII, and PstI) show that in sheep a single chromosome normally bears two nonallelic alpha-globin genes positioned at the same distance as in goat. Variant digestion patterns with enzymes that cleave outside (BamHI and HindIII) and within (EcoRI) the alpha-globin loci allowed us to infer that chromosomes with different numbers of alpha-globin loci are also present in sheep. In particular, in the 60 sheep considered, four individuals were heterozygous (alpha alpha/alpha alpha alpha) and one was homozygous (alpha alpha alpha/alpha alpha alpha) for chromosomes with three loci and one individual was heterozygous for a chromosome with four loci (alpha alpha/alpha alpha alpha alpha). This variation in the number of copies of alpha-globin loci can be explained by means of unequal crossovers.

Animals↗
PubMed1986

A DNA probe detecting multiple haplotypes of the human Y chromosome.

We have characterized a DNA probe (49f) that detects about 15 Y-specific TaqI bands corresponding to a low-copy number sequence. Five of these bands, each representing a single DNA fragment, can either be present, absent, or variable in length. Familial segregation studies have shown that the variations of these fragments are inherited in a Mendelian fashion and strictly Y-linked. A survey of 44 male individuals indicated that the five variable TaqI fragments detected by probe 49f can be considered as five independent allelic series. Each series represents the different and mutually exclusive allelic forms observed for a single DNA fragment. A total of 16 haplotypes, each defined by a different combination of the various forms of each of these five restriction fragment length polymorphisms, were observed among the 44 scored individuals. These TaqI restriction polymorphisms are not observed with other restriction digests and have therefore been attributed to point mutations. The five polymorphic fragments map to Yq11, a region that does not recombine with the X chromosome and are therefore not redistributed. This implies that an apparently independent reassortment of one of these series with respect to the others can be explained only on the basis of mutations that occurred several times (or reverted) during evolution of the Y chromosome. However, an examination of the different combinations of two or more allelic series suggests that some alleles are not randomly distributed and raises the possibility of establishing a genealogy of the human Y chromosome.

Alleles↗
PubMed1988

The long terminal repeat of feline endogenous RD-114 retroviral DNAs: analysis of transcription regulatory activity and nucleotide sequence.

Six cloned 5' long terminal repeat (LTR) and adjoining cellular DNA regions of partially deleted feline endogenous RD-114 proviral loci were linked to the chloramphenicol acetyltransferase (CAT) gene and assayed for their ability to promote transient CAT expression. One endogenous LTR (clone CRL-3) and the LTR from an infectious RD-114 provirus, EX-LTR, were capable of actively expressing the CAT gene. DNA sequence comparison of these LTRs with an inactive endogenous LTR (CR-1) revealed extensive homology in all regions except in the 5' half of U3. The homologous portion contained transcriptional regulatory sequences including CAT, TATA, polyadenylation signal boxes and an octamer enhancer, which is rarely seen in retroviruses. Variations in the 5' half of U3 were primarily due to insertions and deletions. A major difference was in number of copies and integrity of tandemly repeated sequences. EX-LTR contained two pairs of tandem direct repeats, while the two endogenous LTRs contained different deletions of repeated sequences. DNA sequence data also revealed that the primer binding site for RD-114 loci was complementary to a glycine tRNA isotype, the use of which is distinct from any other known retrovirus. An analysis of the steady state RNA levels in T-lymphoid cell lines showed that at least three different incomplete proviral transcripts and their spliced products made up the majority of expressed RD-114 mRNA, and further demonstrated that partially deleted proviral loci have the potential to be transcriptionally vigorous in certain feline cell types.

Acetyltransferases↗
PubMed1992

5S ribosomal gene clusters in wheat: pulsed field gel electrophoresis reveals a high degree of polymorphism.

The long-range structure of 5S rRNA gene clusters has been investigated in wheat (Triticum aestivum L.) by means of pulsed field gel electrophoresis. Using aneuploid stocks, 5S rRNA gene clusters were assigned to sites on chromosomes 1B, 1D, 5B and 5D. Cluster sizes were evaluated and the copy number of 5S DNA repeats was estimated at 4700-5200 copies for the short repeating unit (410 bp) and about 3100 copies for the long repeat (500 bp) per haploid genome. A comparison of wheat cultivars revealed extremely high levels of polymorphism in the 5S rRNA gene clusters. With one restriction enzyme digest all varieties tested gave unique banding patterns and, on a per fragment basis, 21-fold more polymorphism was detected among cultivars for 5S DNA compared to standard restriction fragment length polymorphisms (RFLPs) detected with single copy clones. Experiments with aneuploid stocks suggest that the 5S rRNA gene clusters at several chromosomal sites contribute to this polymorphism. A number of previous reports have shown that wheat cultivars are not easily distinguished by isozymes or RFLPs. The high level of variation detected in 5S rRNA gene clusters therefore offers the possibility of a sensitive fingerprinting method for wheat. 5S DNA and other macro-satellite sequences may also serve as hypervariable Mendelian markers for genetic and breeding experiments in wheat.

Chromosomes↗
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↗
PubMed1988

Comparative study of molecular characteristics of X and Y nucleolar organizers of various lines of Drosophila melanogaster.

We have determined for the X chromosomes of 10 laboratory strains and the Y chromosomes of 4 of them both the total number of ribosomal units and the relative percentages of uninterrupted (ins-), type 1 (ins1: with distinction between small ins1S and large ins1L) and type 2 (ins2) interrupted ribosomal units. These studies were made with the DNA extracted from third instar larval diploid tissues (brains and imaginal discs) of X/X female lines or XNO-/Y male lineages (devoid of X ribosomal genes) whose members possess copies of the same initial X or Y chromosome. Between the X chromosomes as well as the Y chromosomes an approximately equal to 2-fold variation was observed in the total number of ribosomal genes: from approximately equal to 200 to 420 for the X chromosomes and from approximately equal to 150 to 330 for the Y ones. The Y chromosomes are devoid of insertion 1 interrupted units, but one can observe some variation in the percentage and hence the absolute numbers of uninterrupted and insertion 2 interrupted units. Among the X chromosomes a very large variation exists between the percentage and absolute number of all the ribosomal unit types; it is to be noted especially that the number of uninterrupted units, which are the only kind of ribosomal genes actively transcribed, can vary from about 20 to 140 without any differences in the development of the different strains.

Animals↗
PubMed1986

Studies on host-virus genome relationship in Epstein-Barr virus immortalized lymphoblastoid cell lines.

Five human lymphoblastoid cell lines immortalized in vitro with the B95-8 EBV strain, chosen to have a low number of copies of EBV genome, were examined to detect variations in electrophoretic mobility of viral restriction fragments and in the karyotype. Patterns of mobility detected with different viral probes are always the same as those obtained with fragments from purified virus-plasmidic DNA, with one exception. This "non-plasmidic" pattern occurs with a probe containing the termini of the linear virion DNA and consists in an increase of the molecular weight and in the appearance of more than one band. Cytogenetic studies carried on the same cell populations used as source of DNA, early after immortalization, showed a diploid modal chromosome number and no G banding rearrangements.

Cell Line↗