Intragenomic length variation of the ribosomal DNA intergenic spacer in a malaria vector,Anopheles sinensis

We determined the complete sequences of six size variants of intergenic spacer (IGS) region from one individual of the malaria vector mosquito species, Anopheles sinensis. All six size variants observed in this study show almost the same basic primary structure in which three repeat regions (A, B, and C) are interspersed by highly conserved nonrepeating sections. In contrast to the well-ordered subrepeating patterns found in A and C, the repeat region B displays extremely variable and complicated profiles in the number and arrangement of subrepeat units among different size classes. It is apparent that the prominent level of length difference in the repeat regions B and C is responsible for the intragenomic length variations of the IGS molecule observed in the present study. High level of sequence homology and regularly arranged repeating pattern of 11 to 14 bp motif sequences harbored within the B repeat region allow us to consider that these motif sequences may be associated with their potential role as a recombination site. Compared to those previously published in other mosquito species, the IGS of A. sinensis showed a very unique structural format in subrepeat patterns of the IGS region. This result suggests that the structure and sequence profiles of the IGS region would provide useful information for the exploitation of a convenient molecular marker to identify morphologically complicated species complex and to characterize the genetic variation of population. This suggestion is far from being conclusive at present, but a further genetic study will bring more compelling evidences for this pending issue.     

Evolution of a VNTR located within the promoter region of the thiopurine methyltransferase gene: inferences from population and sequence data

The promoter region of the human thiopurine methyltransferase (TPMT) gene contains a variable number of tandem repeats (VNTR) with three kind of motifs (A, B, and C) differing by the length of the unit core and nucleotide sequence. We have studied the structural variation within the VNTR alleles in two human populations and in samples from gorillas and chimpanzees. In humans, no intermingling of motifs was detected within the VNTR, and the sequences of the three core motifs remained remarkably unchanged, differences between alleles corresponding essentially to variations in the number of A and B repeats. The variation pattern in humans is consistent with an interpretation in which two contiguous genetic units (repeats A and B) behave evolutionarily according to the stepwise mutation model, as inferred from the population distribution profiles and from the molecular phylogenetic relationships among the VNTR alleles. However, the observation of a strong negative correlation between the numbers of A and B repeats also suggests that the regularity and/or independence of the mutational process has been disrupted to some extent by interactions between the A and B stretches. Selective pressure (the VNTR plays some role, although minor, in the TPMT function) or biased mutation are possible explanations. In gorillas and chimpanzees, several A-, B-, or C-like motifs were detected, but their arrangement within the VNTR alleles did not followed the regular pattern registered in humans and, particularly for the B-like motifs, a considerable sequence hypervariability was registered. Furthermore, the structural differences among non-human alleles were sufficiently numerous to render more plausible the assumption of the infinite allele model.     

Evolution of exon 1 of the dopamine D4 receptor (DRD4) gene in primates

The dopamine D4 receptor (DRD4) gene exhibits a large amount of expressed polymorphism in humans. To understand the evolutionary history of the first exon of DRD4-which in humans contains a polymorphic 12bp tandem duplication, a polymorphic 13bp deletion, and other rare variants-we examined the homologous exon in thirteen other primate species. The great apes possess a variable number of tandem repeats in the same region as humans, both within and among species. In this sense, the 12bp tandem repeat of exon 1 is similar to the 48bp VNTR of exon 3 of DRD4, previously shown to be polymorphic in all primate species examined. The Old World monkeys show no variation in length, and a much higher conservation of amino acid sequence than great apes and humans. The New World monkeys show interspecific differences in length in the region of the 12bp polymorphism, but otherwise show the higher conservation seen in Old World monkeys. The different patterns of variation in monkeys compared to apes suggest strong purifying selective pressure on the exon in these monkeys, and somewhat different selection, possibly relaxed selection, in the apes.     

Complete mtDNA of Meretrix lusoria (Bivalvia: Veneridae) reveals the presence of an atp8 gene, length variation and heteroplasmy in the control region

The complete nucleotide sequence of the mitochondrial genome of the clam Meretrix lusoria (Bivalvia: Veneridae) was determined. It comprises 20,268 base pairs (bp) and contains 13 protein-coding genes, including ATPase subunit 8 (atp8), two ribosomal RNAs, 22 transfer RNAs, and a non-coding control region. The atp8 encodes a protein of 39 amino acids. All genes are encoded on the same strand. A putative control region (CR or D-loop) was identified in the major non-coding region (NCR) between the tRNA^Gly and tRNA^Gln. A 1087 bp tandem repeat fragment was identified that comprises nearly 11 copies of a 101 bp motif and accounts for approximately 41% of the NCR. The 101 bp tandem repeat motif of the NCR can be folded into a stem–loop secondary structure. Samples of eight individuals from Hainan and Fujian provinces were collected and their NCR regions were successfully amplified and sequenced. The data revealed a highly polymorphic VNTR (variable number of tandem repeats) associated with high levels of heteroplasmy in the D-loop region. The size of the CR ranged from 1942 to 3354 bp depending upon the copy number of the repeat sequence.     

猪PAC克隆的微卫星DNA分离研究

利用(CA)8核心序列,设计锚定引物,采用直接测序法,从单个猪PAC克隆中分离到一个新微卫星DNA。根据该微卫星DNA的侧翼序列,设计了专一引物,在8个猪种40个个体中检测到3个等位基因,片段长度分别为305 bp、307 bp和309 bp。3种等位基因纯合子个体的PCR产物序列分析表明,这3种等位基因分别有12、13和14次CA双核苷酸重复。 Abstract:A novel microsatellite DNA was isolated from a single porcine PAC clone by sequencing the PAC clone directly with (CA)n repeat motif anchored primer.The specific primer pairs flanking the (CA)n repeat region were used to amplify the genomic DNA of 40 individuals from 8 pig breeds,which detected three alleles with the fragment length of 305 bp,307 bp and 309 bp.The PCR product sequencing results of homozygous animals representing three alleles revealed that those three alleles contained 12,13 and 14 CA dinucleotide repeats respectively.     

The complete mitochondrial genome of the taimen, Hucho taimen, and its unusual features in the control region

The whole mitochondrial genome of Hucho taimen was firstly sequenced and characterized. The genome is 16,833 bp in length and contains 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs, and a noncoding control region. Twelve protein-coding genes on the heavy strand showed that the content of A+T was higher than that of G+C, whereas the nd6 protein-coding gene on the light strand displayed an opposite pattern. We described the secondary structure of the origin of light strand (oriL) replication and found that the conserved 5'-GCCGG-3' sequence motif is variable in H. taimen and some other salmonids. We conclude that the control region is variable in length and represents the high A+T content, compared with other mitochondrial control regions available in Salmonidae and other non-salmonids. Additionally, another interesting feature of H. taimen mitogenome is that a T-type mononucleotide microsatellite and an 82 bp tandem repeat were identified in the control region.     

Complete mitochondrial genome of Oxuderces dentatus (Perciformes, Gobioidei)

The complete mitochondrial genome (mitogenome) of Oxuderces dentatus was determined first. The genome was 17,116?bp in length and consisted of 13 protein-coding genes, 22 tRNA genes, 2 ribosomal RNA genes, and 2 main non-coding regions [the control region (CR) and the origin of the light strand replication], the gene composition and order of which was similar to most other vertebrates. The overall base composition of the heavy strand was T 27.9%, C 26.8%, A 30.2%, and G 15.1%, with a slight A+T bias of 58.1%. In addition to the discrete and conserved sequence blocks, unusual long tandem repeat unit (three 150-bp tandem repeat units and an incomplete copy of 146?bp) was also detected within CR. This mitogenome sequence data would play an important role in population genetics and phylogenetic analysis of the Gobioidei.     

Evolution and phylogenetic information content of the ribosomal DNA repeat unit in the Blattodea (Insecta)

The organization, structure, and nucleotide variability of the ribosomal repeat unit was compared among families, genera, and species of cockroaches (Insecta:Blattodea). Sequence comparisons and molecular phylogenetic analyses were used to describe rDNA repeat unit variation at differing taxonomic levels. A reverse similar 1200 bp fragment of the 28S rDNA sequence was assessed for its potential utility in reconstructing higher-level phylogenetic relationships in cockroaches. Parsimony and maximum likelihood analyses of these data strongly support the expected pattern of relationships among cockroach groups. The examined 5' end of the 28S rDNA is shown to be an informative marker for larger studies of cockroach phylogeny. Comparative analysis of the nucleotide sequences of the rDNA internal transcribed spacers (ITS1 and ITS2) among closely related species of Blattella and Periplaneta reveals that ITS sequences can vary widely in primary sequence, length, and folding pattern. Secondary structure estimates for the ITS region of Blattella species indicate that variation in this spacer region can also influence the folding pattern of the 5.8S subunit. These results support the idea that ITS sequences play an important role in the stability and function of the rRNA cluster.     

The complete mitochondrial genome sequence of Sogatella furcifera (Horváth) and a comparative mitogenomic analysis of three predominant rice planthoppers

The white-backed planthopper (WBPH), Sogatella furcifera (Horváth), is one of the most destructive pests of rice crops in many Asian countries. Using long-PCR and shotgun library methods, we sequenced the entire mitochondrial genomes (mt-genomes) of two WBPH individuals. Total lengths of the mt-genome of the two WBPH individuals were 16,612 bp and 16,654 bp with an identical AT content of 76.19%. Among the 13 protein coding genes (PCGs), only nad5 used an atypical initiation codon GTG. Most of the tRNA genes had the typical cloverleaf secondary structure except that the dihydrouridine (DHU) arms in two trnS genes and the TΨC arm of trnG gene did not form a stable stem-loop structure. Similar to the brown planthopper (BPH), Nilaparvata lugens (Stål), and the small brown planthopper (SBPH), Laodelphax striatellus (Fallén), some extraordinary features were observed in the WBPH mt-genome. These include similar gene rearrangement pattern, unusually short length of the atp8 gene and variable numbers of tandem repeat (VNTR) structure in control region. Interestingly, the same tandem repeat unit with stable secondary structure appeared in two different planthoppers, WBPH and SBPH, which belong to two different genera of the Delphacidae. This peculiar feature provides a direct evidence for the close relationship between the two planthoppers and updates our understanding of the evolutionary characteristics of mitochondrial control region. Comparison with two other predominant rice planthoppers (BPH and SBPH) revealed that different PCGs of mitochondria exhibit different evolutionary patterns.     

中国人端粒酶催化亚基(hTERT)基因第六内含子VNTR多态性研究

端粒酶的激活在肿瘤发生、衰老以及细胞永生化过程中起重要作用,端粒酶催化亚基(hTERT)的表达是诱导端粒酶活性的限制性步骤,hTERT的表达受到复杂的调控。hTERT基因组全长40kh,包含16个外显子及15个内含子,其中,在第2和第6内含子中各存在2个可变数目串联重复(VNTR)多态性序列(VNTR2—1st、VNTR2—2nd以及VNTR6—1st和VNTR6—2nd),在第12内含子存在另一个单态性串联重复序列。通过对北京地区210例汉族健康人群hTERT基因第6内含子中36-bpVNTR6—1st的多态性进行调查研究．结果在调查人群中观察到18、20、21、22、23、26和35次重复共7种等位基因型以及14种基因型。基因型频率分布符合Hardy—Weinberg平衡。与韩国浦山地区调查人群类似,20、22及35次重复为最常见的等位基因型,这3种等位基因型频率占调查人群总数的94．76％。除了35次重复等位基因型频率与浦山地区人群存在差异外,其他基因型频率差异不显著。此外,除了在部分重复22次的等位基因型中VNTR5′端与浦山地区人群相同外,其他等位基因型中,北京地区汉族人VNTR6—1st5′端均存在53bD插入片段,显示出不同人种vNTR6—1st周边序列的差异性。北京地区汉族正常人群hTERT VNTR基因多态性资料为研究多态性与肿瘤或衰老的相关性提供了资料。     

Complete mitochondrial genome of the mudskipper Boleophthalmus pectinirostris (Perciformes, Gobiidae): repetitive sequences in the control region

The mudskipper, Boleophthalmus pectinirostris (Perciformes, Gobiidae), is an amphibious gobioid fish. In this paper, the complete mitochondrial genome of B. pectinirostris was firstly determined. The mitogenome (17,111 bp) comprises 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and 1 putative control region. 130-bp tandem repeat was identified in the control region, which was almost identical among the 10 individuals examined, and three different frequencies of the repeat unit (five, six or seven) were found among these individuals.     

Identification of a variable number tandem repeat region in the human T cell receptor alpha-delta (TCRAD) locus

A number of different polymorphisms have been observed in coding as well as in non-coding regions of T cell receptor (TCR) genes. We report the identification and characterization of a highly polymorphic locus in the 3′ noncoding region of the human T cell receptor α/δ (TCRAD) on chromosome 14. In 202 unrelated individuals, ten different alleles were distinguished by polymerase chain reaction (PCR) and a heterozygosity rate of 64% was calculated. Sequence analysis revealed that this polymorphic region consists of 10 bp imperfect repeat units and represents a variable number tandem repeat region (VNTR). Stable Mendelian inheritance of this novel polymorphic marker was proven in four families. The localization of this VNTR polymorphism in the TCRAD locus should make it a useful system for linkage analysis in immunological disorders with a known role of TCRAD. Received: 19 February 1996 / Revised: 27 March 1996     

Relationship of micro-and minisatellites in the human genome

Micro-and minisatellites constitute an essential part of DNA with low sequence complexity and perform a number of important functions. The TandemSWAN program was used to search the human genome for tandem repeats with a length of a repeated unit to 70 bp, including repeats with a large number of nucleotide substitutions. It was shown that, for a significant fraction of the program-found minisatellites with a repeat unit length less than 25 bp, a shorter repeated motif can be discerned in this sequence, which is often similar to the sequence of microsatellites occurring widely in the human genome. A model of hierarchical origin of minisatellites in the human genome was proposed.     

Population genetic structure of the migratory rice leaf roller, Cnaphalocrocis medinalis (Lepidoptera: Pyralidae), inferred from the mitochondrial A+T-rich region and nuclear ITS2 sequences

The population genetics of the migratory rice leaf roller, Cnaphalocrocis medinalis (Lepidoptera: Pyralidae), was characterized using the maternally inherited mitochondrial A+T-rich region and bi-parentally inherited nuclear internal transcribed spacer 2 (ITS2). One hundred and eighty-seven specimens of the rice leaf roller collected from 13 Korean and Chinese localities revealed 94 A+T-rich region haplotypes, ranging in sequence length from 339 to 348 bp and 129 ITS2 sequence types, ranging from 444 to 450 bp, with maximum sequence divergences of 4.55 and 4.43%, respectively. The finding of almost no significant F(ST), even among Chinese and Korean localities, except for one Chinese island population (ITS2 only), and the finding of genetic variance principally at the within-population level indicate the genetic structure characteristics of a migratory insect that is well connected among populations due to high gene flow. Detection of significant F(ST) estimates of one offshore island population in China (Haikou) compared to most others only by ITS2 rather than by the mitochondrial A+T-rich region, as well as the somewhat higher degree of genetic differentiation seen on ITS2, suggest the importance of female dispersal. Structural analysis of the A+T-rich region revealed a poly-T stretch (10-16 bp), a microsatellite-like AT repeat (10-14 repeats), and a 5-bp long-motif "ATTTA". The typical 5-bp long conserved motif sequence (ATAGA) previously detected in other lepidopterans was found to be ATAG in the C. medinalis A+T-rich region.     

Rapid diagnosis of Miller-Dieker syndrome and isolated lissencephaly sequence by the polymerase chain reaction

Summary Probe YNZ22 (D17S5) is a highly polymorphic, variable number tandem repeat (VNTR) marker previously shown to be deleted in all patients with the Miller-Dieker syndrome (MDS) but not in patients with isolated lissencephaly sequence (ILS). Primers were constructed to the unique sequence flanking the polymorphic, repetitive region of YNZ22 for amplification by the polymerase chain reaction (PCR). Analysis of 118 normal individuals revealed 12 alleles (differing in copy number of a 70-bp repeat unit) ranging in size from 168 to 938 bp. A retrospective study of eight MDS and six ILS patients was consistent with Southern blot analysis in all cases except one. In the latter, a very large allele (12 copies of the repeat unit) in a patient and her mother failed to amplify on initial attempts, but was successfully amplified by reducing the concentration of genomic DNA used in the reaction. Prospective studies on two MDS and five ILS patients were successfully performed and confirmed in all cases by Southern blot analysis. From the total sample, restriction fragment length polymorphism (RFLP) analysis was fully informative in four of ten MDS patients and showed a deletion in all four cases. Nine of eleven ILS patients were heterozygous and therefore not deleted for YNZ22. Development of primers for additional polymorphic markers in the Miller-Dieker region will lead to a rapid PCR-based diagnostic approach for all MDS and ILS patients. PCR typing of YNZ22 will also facilitate use of this marker in other applications, including genetic linkage, paternity and forensic studies, and analysis of loss of heterozygosity in tumors.     

Allele Frequency of VNTR Locus D1S80 Observed in Denizli Province of Turkey

The variable numbers of tandem repeats (VNTR) locus D1S80, located on chromosome 1 (1p35-36), has a repeat unit 16 bp in length, and different numbers of these repeat units have been observed for populations of different origins and ethnicity. We used a molecular identification method based on capillary electrophoresis separation to analyze D1S80 locus polymorphism among 74 subjects from Denizli province, Turkey, finding an amplified fragment length size of 379–635 bp. Allele repeat numbers were deduced from these sizes and sequence comparison. The most common alleles were repeat units 24 (34.3%) and 18 (22.4%), with frequencies of 0.414 and 0.207, respectively. Other alleles were 25 (7.86%), 28 (5.71%), 22 (4.25%), and 29 (2.86%). The allele with 23 repeat units was not observed. Results were in Hardy–Weinberg linkage disequilibrium. Observed heterozygosity was 0.614, and expected heterozygosity was 0.787. Theta(k) value was 4.86 (95% confidence interval limits). Capillary electrophoresis is a powerful approach for accurate identification of VNTR loci, especially for low base pair units like D1S80, for prenatal diagnosis, linkage analysis, forensic identification, paternity testing, anthropological research, and phylogenetic studies.     

Ribosomal DNA Intergenic Spacer of the Swimming Crab, Charybdis japonica

We have determined the full sequence of the ribosomal DNA intergenic spacer (IGS) of the swimming crab, Charybdis japonica, by long PCR for the first time in crustacean decapods. The IGS is 5376 bp long and contains two nonrepetitive regions separated by one long repetitive region, which is composed mainly of four subrepeats (subrepeats I, II, III, and IV). Subrepeat I contains nine copies of a 60-bp repeat unit, in which two similar repeat types (60 bp-a and 60 bp-b) occur alternatively. Subrepeat II consists of nine successive repeat units with a consensus sequence length of 142 bp. Subrepeat III consists of seven copies of another 60-bp repeat unit (60 bp-c) whose sequence is complementary to that of subrepeat I. Immediately downstream of subrepeat III is subrepeat IV, consisting of three copies of a 391-bp repeat unit. Based on comparative analysis among the subrepeats and repeat units, a possible evolutionary process responsible for the formation of the repetitive region is inferred, which involves the duplication of a 60-bp subrepeat unit (60 bp-c) as a prototype. Received: 13 April 1999 / Accepted: 2 August 1999     

Relationship between micro- and minisatellites in the human genome

Micro- and minisatellites constitute an essential part of DNA with a low sequence complexity and carry several important functions. A search for tandem repeats in the human genome with a length of a repeat unit of up to 70 bp, including repeats with a great number of nucleotide substitutions, has been performed using the TaadeaSWAN program. It was shown that, for a considerable number of minisatellites with the length of the repeating unit of less than 25 nt, a shorter repeating motif can be distinguished in the sequence of this repeat, which often is similar to the sequence of minisatellites widely occurring in the human genome. A model of hierarchic origination of minisatellites in the human genome is suggested.