Polymorphic simple GATA/GACA repeats in plant genomes.
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Base Sequence↗
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Base substitutions, length differences and DNA strand asymmetries in the human G gamma and A gamma fetal globin gene region.
We have studied differences arising subsequent to the 5 kilobase pair (kb) duplication that led to the human G gamma and A gamma fetal globin genes. The local occurrence of base substitutions in the duplicated 5 kb region correlates positively with the local AT base pair content. This correlation also occurs in two mouse beta-globin genes and in two mouse immunoglobulin genes. The relationship is valid for transcribed or nontranscribed DNA and for DNA that contains only coding sequences. Length differences in the fetal globin duplicated regions correlate positively with the occurrence of short direct repeats of greater than or equal 5 base pairs. Path analysis of the interrelationships of base composition, base substitutions, repeats and length differences provides an integrated view of the relative effects on chromosomal changes of these variables and of selection. The distributions along the chromosome of simple sequences and of base compositions show highly significant local asymmetries between the transcribed and nontranscribed strands of the DNA, which permit us to divide the fetal globin gene region into chromosomal domains. Comparable domains are present in DNA from other sources, including the mammalian viruses SV40 and polyoma virus strain A-2 in which some of the domains appear related to discrete functions.
Animals↗
[A detection for replication error (RER) and LOH by microsatellite analysis in human lung cancer].
Recently, it has been considered that the pleomorphic changes in the microsatellite areas, which are said to exist at 50 to 100 thousand places on the human chromosome, are the results of abnormality in the repairing mechanism in cancer cells, and active attempts are now being made to detect such changes using PCR. In the DNA of cancer tissue where the repairing function has decreased, sequence repeat different in length from that of the DNA of normal tissue is observed. Such abnormal repeat shows itself as replication error (RER). As method of detecting RER, PCR was hitherto performed using RI-labeled primer, and after electrophoresis, detection was made by autoradiography. In order to obtain a reproducible result in a simple way, we attempted to detect by RER with auto-sequencer in 60 patients with lung cancer. As a result, we obtained data with a high sensitivity and objectivity. Therefore, a report is made on this study of ours.
Base Sequence↗
A review of the use of ribosomal DNA (rDNA) to differentiate among cryptic Anopheles species.
Cryptic species complexes are groups of closely related species that are difficult or impossible to distinguish by morphological traits. These complexes are known from a wide variety of arthropods and are common among the well-studied, medically-important insects. For example, many of the anopheline vectors of malaria parasites are members of cryptic species complexes. Complexes typically include both vector and non-vector species, and two or more member species are often found sympatrically. Until the late 1950, only two such Anopheles complexes were known, the A. gambiae complex from Africa and the A. maculipennis complex from Europe. Today, dozens of Anopheles cryptic species complexes are recognized, and accumulating evidence suggests that most important malaria vectors are likely to be members of such complexes. A variety of methods have been developed for identifying the species of individual specimens from these complexes, although until recently only those based on species-specific allozymes and polytene chromosome inversions were widely used. The limitations inherent in these methods have been circumvented with DNA-based procedures, which are especially useful because both sexes and all developmental stages can be identified, and DNA can be recovered from samples stored by a wide variety of simple methods. Several DNA-based identification techniques have been developed, including hybridization assays based on species-specific repeat sequences, and diagnostic PCR fragments produced either by the use of random PCR primers or by amplifying DNA with primers based on known species-specific sequences. In this review we discuss the relative marks of different methods of cryptic species identification, with emphasis on the use of ribosomal DNA as a target for species-diagnostic PCR assays.
Animals↗
Chromosome structure: DNA nucleotide sequence elements of a subset of the minichromosomes of the protozoan Trypanosoma brucei.
The genome of the protozoan Trypanosoma brucei contains a set of about 100 minichromosomes of about 50 to 150 kb in size. The small size of these chromosomes, their involvement in antigenic variation, and their mitotic stability make them ideal candidates for a structural analysis of protozoan chromosomes and their telomeres. We show that a subset of the minichromosomes is composed predominantly of simple-sequence DNA, with over 90% of the length of the minichromosome consisting of a tandem array of 177-bp repeats, indicating that these molecules have limited protein-coding capacity. Proceeding from the tip of the telomere to a chromosome internal position, a subset of the minichromosomes contained the GGGTTA telomere repeat, a 29-bp telomere-derived repeat, a region containing 74-bp G + C-rich direct repeats separated by approximately 155 bp of A + T-rich DNA that has a bent character, and 50 to 150 kb of the 177-bp repeat. Several of the minichromosome-derived telomeres did not encode protein-coding genes, indicating that the repertoire of telomeric variant cell surface glycoprotein genes is restricted to some telomeres only. The telomere organization in trypanosomes shares striking similarities to the organization of telomeres and subtelomeres in humans, yeasts, and plasmodia. An electron microscopic analysis of the minichromosomes showed that they are linear molecules without abnormal structures in the main body of the chromosome. The structure of replicating molecules indicated that minichromosomes probably have a single bidirectional origin of replication located in the body of the chromosome. We propose a model for the structure of the trypanosome minichromosomes.
Animals↗
Low frequency of microsatellite instability in sporadic breast cancer.
In some tumors, defects in mismatch repair enzymes lead to errors in the replication of simple nucleotide repeat segments. This condition is commonly known as microsatellite instability (MSI) because of the frequent mutations of microsatellite sequences. Although the MSI phenotype is well recognized in some colon, gastric, pancreatic, and endometrial cancers, reports of MSI in breast cancer are inconsistent. The purpose of this study was to determine the presence of MSI in breast cancer and to correlate its occurrence with clinicopathological parameters, including expression of estrogen and progesterone receptors. We have analyzed the status of 10 different microsatellite loci (mono and dinucleotide repeats). Mmicrosatellite size patterns and LOH were compared in 88 paired breast-cancer/peripheral-blood DNA samples. Fluorescent polymerase chain reaction (PCR) for typing microsatellites coupled with DNA fragment analysis in an automated DNA sequencer was applied. Microsatellite instability in at least two microsatellite markers was observed in 6 out of 88 (7%) of the cases, all beloging to stage II or III. LOH was found in 48/88 (55%) of the cases. Five of the six cases with MSI also had LOH in other markers different from those of MSI. These MI and LOH data were analysed using a range of clinicopathological parameters, no correlation between MSI and histopathological characteristics were found. A significant correlation was observed between MSI and negative expression of both estrogen and progesterone receptors (p<0.02), indicating a possible relatioship between specific genetic changes at these microsatellite regions and hormonal deregulation in the progresion of breast cancer.
Adult↗
Development and laboratory evaluation of a polymerase chain reaction for monitoring Schistosoma mansoni infestation of water.
A sensitive and specific detection of cercariae of human schistosomes is required for better definition of risk of infection. In the present study, we have developed a polymerase chain reaction (PCR) assay for the detection of cercariae of Schistosoma mansoni in water. A simple DNA preparation was adapted for this purpose, and PCR primers were designed based on the 121-basepair highly repeated sequence we previously identified in the genome of S. mansoni. The PCR assay detected as little as 10(-6) ng of S. mansoni DNA, and the high sensitivity enabled the detection of a single cercaria. For trapping of cercariae we adapted a filtration apparatus previously used for separating schistosome eggs from turbid enzymatic digests of tissues. A single cercaria could be detected in repeated tests of water filtrates. Since the target DNA is tandemly arranged, a ladder pattern of the PCR products was demonstrated. A direct relationship was demonstrated between the number of ladder bands of the amplification products, and DNA concentration or number of cercariae. The feasibility of semiquantitation of schistosome larvae in natural water was thus suggested. The potential of the procedures described here for epidemiologic studies is discussed.
Animals↗
A polymerase chain reaction assay for detecting snails infected with bilharzia parasites (Schistosoma mansoni) from very early prepatency.
In the present study, we adapted a polymerase chain reaction (PCR) assay, previously shown by us to be very sensitive for detecting cercariae in water, for the sensitive detection of Schistosoma mansoni DNA in infected snails from early prepatency. Polymerase chain reaction primers were designed based on the 121-basepair highly repeated sequence we previously identified in the genome of S. mansoni. The DNA was prepared from the snails by a simple alkaline extraction procedure, and the PCR assay enabled a clear differentiation between infected and normal snails. Infected snails were detected as early as one day after penetration of a single miracidium. The high sensitivity of the test enabled identification of a single infected snail even when its DNA was pooled with material from up to 99 uninfected snails, thus demonstrating the possibility of mass diagnosis in pools of snails. The assay has the potential for large-scale determination of prepatent infection prevalence in snails, thus offering new possibilities for the evaluation of schistosomiasis transmission and for schistosomiasis control, as discussed.
Animals↗
Yeast DNA repair protein RAD5 that promotes instability of simple repetitive sequences is a DNA-dependent ATPase.
The RAD5 gene of Saccharomyces cerevisiae functions in postreplication repair of ultraviolet damaged DNA, and interestingly, it also has a role in increasing the instability of simple repetitive sequences in the genome. In contrast to DNA mismatch repair genes which function in maintaining constant length of repeat sequences, RAD5 promotes alterations in the length of repeat sequences. In this work, we purify the RAD5 protein to near homogeneity from yeast cells and show that it is a single-stranded DNA-dependent ATPase. The possible roles of RAD5 ATPase in postreplication gap filling and in increasing the incidence of length alterations of repeat sequences are discussed.
Adenosine Triphosphatases↗
The sequence and organization of Ddp2, a high-copy-number nuclear plasmid of Dictyostelium discoideum.
The complete nucleotide sequence of the plasmid Ddp2 found in the nucleus of the simple eukaryote Dictyostelium discoideum is reported. This 5852-bp plasmid contains a 2661-bp open reading frame (ORF), named the "Rep gene," and 501-bp imperfect inverted repeats. A 1762-bp section of Ddp2, which includes one of the 501-bp repeat sequences, could be deleted without abolishing extrachromosomal replication. Deletion of the second 501-bp repeat, or interruption of the Rep gene, removed the ability to replicate extrachromosomally. We suggest that Ddp2 encodes a protein, "REP," that positively regulates replication initiation, a regulatory mechanism different from that of the yeast 2 mu plasmid which also possesses inverted repeat sequences. Ddp2 has a structure similar to that of plasmid pDG1, found in an unidentified isolate of Dictyostelium, with a similar sized ORF and inverted repeats. A common evolutionary origin is suggested by considerable sequence homology between the ORFs of pDG1 and Ddp2.
Amino Acid Sequence↗
Physical and genetic mapping of cloned ribosomal DNA from Toxoplasma gondii: primary and secondary structure of the 5S gene.
The ribosomal DNA (rDNA encoding rRNA) of the obligately intracellular protozoan parasite, Toxoplasma gondii, was identified, cloned, physically mapped, its copy number determined, and the 5S gene sequenced. Using total RNA as a probe, a collection of recombinant lambda phages containing copies of rDNA were isolated from a lambda 2001 tachyzoite genomic library. Northern gel hybridization confirmed specific homology of the 7.5-kb rDNA unit, subcloned into pTZ18R, to T. gondii rRNA. The mapped rDNA found in pTOX1 contained small ribosomal subunit (SS; 18S)- and large ribosomal subunit (LS; 26S)-encoding genes localized using intragenic heterologous probes from the conserved sequences of the SS (18S) and LS (28S) Xenopus laevis genes. the physical mapping data, together with partial digestion experiments and Southern gel hybridization, confirmed a 7.5-kb rDNA unit arranged in a simple head-to-tail fashion that is tandemly repeated. We estimated the rDNA repeat copy number in T. gondii to be 110 copies per haploid tachyzoite genome. Parts of the SS gene and the complete 5S gene were sequenced. The 5S gene was found to be within the rDNA locus, a rare occurrence found only in some fungi and protozoa. Secondary-structure analysis revealed an organization remarkably similar to the 5S RNA of eukaryotes.
Animals↗
Simple sequence-length polymorphism analysis.
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Acrylic Resins↗
Extremely complex pattern of microsatellite mutation in the germline of wheat exposed to the post-Chernobyl radioactive contamination.
The molecular structure of rare variants at 13 microsatellite loci found in a population of wheat plants grown for one generation in the heavily contaminated 30 km exclusion zone around the Chernobyl Nuclear Power Plant and in a control population was compared. Evidence for rare alterations (variants) was obtained for all 13 loci, including gain and loss of repeats, as well as the complete loss of microsatellite bands. The ratio between gains and losses among variants in the control group was similar to that in the exposed group. Sequencing of variants at six microsatellite loci found in the exposed population revealed extremely complex pattern of germline mutations, including complete deletions of loci, a bias towards mutations with gains and losses of multiple repeat units, and relatively frequent insertions of DNA of unknown origin. The occurrence of large deletions at two loci may be attributed to direct and inverted repeats sequences located just upstream and downstream of the array. The results of our study also suggest that the majority of mutations within the studied wheat microsatellite loci are represented by gains and losses of multiple repeat units, implying that a simple model of replication slippage cannot account for mutation events at these loci. Our data also support the conclusion that the spectra of spontaneous and radiation-induced mutation in wheat may be similar.
Base Sequence↗
Spontaneous deletions and duplications of sequences in the genome of cowpox virus.
Examination of the genomes of 10 white-pock variants of cowpox virus strain Brighton red (CPV-BR) revealed that 9 of them had lost 32 to 38 kilobase pairs (kbp) from their right-hand ends and that the deleted sequences had been replaced by inverted copies of regions from 21 to 50 kbp long from the left-hand end of the genome. These variants thus possess inverted terminal repeats (ITRs) from 21 to 50 kbp long; all are longer than the ITRs of CPV-BR (10 kbp). The 10th variant is a simple deletion mutant that has lost the sequences between 32 and 12 kbp from the right-hand end of the genome. The limits of the inner ends of the observed deletions (between 32 and 38 kbp from the right-hand end of the CPV-BR genome) appear to be defined by the location of the nearest essential gene on the one hand and the location of the gene that encodes "pock redness" on the other. The genomes of the deletion/duplication white-pock variants appear to have been generated either by single crossover recombinational events between two CPV-BR genomes aligned in opposite directions or by the nonreciprocal transfer of genetic information. The sites where such recombination/transfer occurred were sequenced in four variants. In all of them, the sequences adjacent to such sites show no sequence homology or any other unusual structural feature. The analogous sites at the internal ends of the two ITRs of CPV-BR also were sequenced and also show no unusual features. It is likely that the ITRs of CPV-BR and of its white-pock variants, and probably those of other orthopox-virus genomes, arise as a result of nonhomologous recombination or by random nonreciprocal transfer of genetic information.
Base Sequence↗
Sequence complexity of histone H1 subtypes.
H1 subtypes are involved in chromatin higher-order structure and gene regulation. H1 has a characteristic three-domain structure. We studied the length variation of the available H1 subtypes and showed that the length of the N-terminal and C-terminal domains was more variable than that of the central domain. The N-terminal and C-terminal domains were of low sequence complexity both at the nucleotide and at the amino acid level, whereas the globular domain was of high complexity. In most subtypes, low complexity was due only to cryptic simplicity, which reflects the clustering of a number of short and often imperfect sequence motifs. However, a subset of subtypes from eubacteria, plants, and invertebrates contained tandem repeats of short amino acid motifs (four to 12 residues), which could amount to a large proportion of the terminal domains. In addition, some other subtypes, such as those of Drosophila and mammalian H1t, were only marginally simple. The coexistence of these three kinds of subtypes suggests that the terminal domains could have originated in the amplification of short sequence motifs, which would then have evolved by point mutation and further slippage.
Animals↗
Replication of Mus dunni endogenous retrovirus depends on promoter activation followed by enhancer multimerization.
Mus dunni endogenous virus (MDEV) is an apparently intact retrovirus that normally lies transcriptionally silent in cultured M. dunni cells, but the provirus can be activated by treatment of the cells with hydrocortisone or 5-iodo-2'-deoxyuridine. Sequence analysis of a molecular clone of the replicating virus revealed a simple retrovirus with a chimeric VL30/GALV-like structure. Interestingly, in the region of the long terminal repeat (LTR) that typically contains the retroviral transcription enhancers, we found over six 80-bp repeats with only a single mismatch, indicating that acquisition of the repeats was a recent event. Here we provide evidence for the following model of MDEV activation and replication. The MDEV provirus in M. dunni cells has a chimeric structure similar to that of the molecular clone but has only 1.15 copies of the 80-bp repeat sequence found in the molecular clone. Activating chemicals directly stimulate transcription from the LTR, allowing a low level of virus replication. Copying errors made during reverse transcription allow multimerization of the 80-bp enhancer region, resulting in viruses with higher transcriptional rates and improved fitness, but increased enhancer copy number is likely balanced by the natural instability of retroviral repeats and constraints imposed by virion packaging limits. The resultant population of replicating MDEV is widely heterogeneous, having from 2.15 to 13.15 enhancer repeats in the LTR. These results reveal a novel mechanism for regulation of transcription and replication of an endogenous retrovirus, in terms of both activation of the virus by the steroid hydrocortisone and the large number and variation in enhancer repeats observed.
Animals↗
Tandemly repeated DNA is a target for the partial replacement of thymine by beta-D-glucosyl-hydroxymethyluracil in Trypanosoma brucei.
In the DNA of African trypanosomes a small fraction of thymine is replaced by the modified base beta-D-glucosyl-hydroxymethyluracil (J). The function of this large base is unknown. The presence of J in the silent variant surface glycoprotein gene expression sites and the lack of J in the transcribed expression site indicates that DNA modification might play a role in control of gene repression. However, the abundance of J in the long telomeric repeat tracts and in subtelomeric arrays of simple repeats suggests that J may also have specific functions in repetitive DNA. We have now analyzed chromosome-internal repetitive sequences in the genome of Trypanosoma brucei and found J in the minichromosomal 177-bp repeats, in the long arrays of 5S RNA gene repeats, and in the spliced-leader RNA gene repeats. No J was found in the rDNA locus or in dispersed repetitive transposon-like elements. Remarkably, the rDNA of T. brucei is not organized in long arrays of tandem repeats, as in many other eukaryotes. T. brucei contains only approximately 15-20 rDNA repeat units that are divided over six to seven chromosomes. Our results show that J is present in many tandemly repeated sequences, either at a telomere or chromosome internal. The presence of J might help to stabilize the long arrays of repeats in the genome.
Animals↗
Actin as the generator of tension during muscle contraction.
We propose that the key structural feature in the conversion of chemical free energy into mechanical work by actomyosin is a myosin-induced change in the length of the actin filament. As reported earlier, there is evidence that helical actin filaments can untwist into ribbons having an increased intersubunit repeat. Regular patterns of actomyosin interactions arise when ribbons are aligned with myosin thick filaments, because the repeat distance of the myosin lattice (429 A) is an integral multiple of the subunit repeat in the ribbon (35.7 A). This commensurability property of the actomyosin lattice leads to a simple mechanism for controlling the sequence of events in chemical-mechanical transduction. A role for tropomyosin in transmitting the forces developed by actomyosin is proposed. In this paper, we describe how these transduction principles provide the basis for a theory of muscle contraction.
Actins↗