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Molecular phylogeography of the red deer (Cervus elaphus) populations in Xinjiang of China: comparison with other Asian, European, and North American populations.

To illustrate phylogeography of red deer (Cervus elaphus) populations of Xinjiang, we determined their mitochondrial DNA (mtDNA) control region sequences, and then investigated geographic variations and phylogenetic relationships between Xinjiang populations and other populations from Asia, Europe, and North America. The C. elaphus mtDNA control region shared different copy numbers of tandem repeats of 38 to 43-bp motifs which clearly distinguished the Western lineage from the Eastern lineage of this species in Eurasia. The western lineage comprised the Tarim populations from southern Xinjiang and the European populations, all of which had four copies of the motifs. By contrast, the Eastern lineage consisted of populations from northern Xinjiang (Tianshan and Altai Mountains), other Asian areas (Alashan, Gansu, Tibet, Mongolia, and northeastern China), and North America, all of which shared six copies of the motifs. MtDNA phylogenetic trees showed that there are two major clusters of haplotypes which referred to the Western and Eastern lineages, and that subgroupings of haplotypes in each cluster were congruent with their geographic distributions. The present study revealed that a boundary separating the Western lineage from the Eastern lineage occurs between Tarim Basin and Tianshan Mountains in Xinjiang. Meanwhile, North American populations were genetically closer to those of northern Xinjiang, northeastern China, and Mongolia, supporting that C. elaphus immigrated from northeastern Eurasia to North America through the glacier-induced land-bridge (Beringia) which had formed between the two continents after Late Pleistocene.

Animals↗

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↗

Characterization of the variable-number tandem repeats in vrrA from different Bacillus anthracis isolates.

PCR analysis of 198 Bacillus anthracis isolates revealed a variable region of DNA sequence differing in length among the isolates. Five polymorphisms differed by the presence of two to six copies of the 12-bp tandem repeat 5'-CAATATCAACAA-3'. This variable-number tandem repeat (VNTR) region is located within a larger sequence containing one complete open reading frame that encodes a putative 30-kDa protein. Length variation did not change the reading frame of the encoded protein and only changed the copy number of a 4-amino-acid sequence (QYQQ) from 2 to 6. The structure of the VNTR region suggests that these multiple repeats are generated by recombination or polymerase slippage. Protein structures predicted from the reverse-translated DNA sequence suggest that any structural changes in the encoded protein are confined to the region encoded by the VNTR sequence. Copy number differences in the VNTR region were used to define five different B. anthracis alleles. Characterization of 198 isolates revealed allele frequencies of 6.1, 17.7, 59.6, 5.6, and 11.1% sequentially from shorter to longer alleles. The high degree of polymorphism in the VNTR region provides a criterion for assigning isolates to five allelic categories. There is a correlation between categories and geographic distribution. Such molecular markers can be used to monitor the epidemiology of anthrax outbreaks in domestic and native herbivore populations.

Bacillus anthracis↗

Selective DNA amplification regulates transcript levels in plant mitochondria.

Most plant mitochondrial genomes exist as subgenomic-size fragments apparently due to recombination between repetitive sequences. This leads to the possibility that independently replicating subgenomic domains could result in mitochondrial gene copy number variation. We show, through Southern-blot analysis of both restricted and intact mtDNA, that there are gene-specific copy number differences in the monocot Zea mays. Comparison of two different maize genotypes, B37(N) and B37(T), a cytoplasmic male-sterile strain, reveal fewer gene copy number differences for B37(T) than for B37(N). In contrast to maize, significant gene copy number differences are not detected in the dicot Brassica hirta. We also demonstrate that mitochondrial transcriptional rates in both species are apparently dependent on gene copy number since relative rates determined by run-on analysis are proportional to relative gene copy numbers. Thus a direct relationship exists between plant mitochondrial gene copy number and transcriptional rate.

Blotting, Southern↗

Molecular evolution of a tandemly repeated trnF(GAA) gene in the chloroplast genomes of Microseris (Asteraceae) and the use of structural mutations in phylogenetic analyses.

We sequenced the first ca. 900 bp of the 5'-trnL(UAA)-trnV(UAC)/ndhJ region of the chloroplast DNA of different Microseris accessions in order to resolve homoplasious length variation detected in the trnL(UAA)-trnF(GAA) region. We found two to four tandemly repeated trnF genes in the species of Microseris (Asteraceae, Lactuceae) and two in their sister genus Uropappus. Sequences indicated nonhomologous transitions between two, three, and four trnF genes in different Microseris taxa. Independent origins of similar trnF copy numbers were inferred from a chloroplast phylogeny of Microseris. The taxa involved grow on separate continents, supporting parallel origins of similar length variants. The changes in trnF copy numbers were best explained by interchromosomal recombination with unequal crossing over. The 5' copies of the repeats showed the highest sequence conservation, suggesting that these copies are likely to be functional trnF genes, whereas the other ones probably represent pseudogenes. Our results show that length polymorphisms accumulate once a duplicated sequence has become incorporated. Due to parallel gains of similar trnF copy numbers, homoplasious length variation was introduced into the data matrix. The data demonstrate that length polymorphisms cannot be used as indicators for phylogenetic distance unless they can be analyzed at the sequence level.

Base Sequence↗

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↗

[Forensic DNA analysis--past and future].

Since the introduction of DNA polymorphism analysis techniques to forensic science, forensic identification research has made radical, astonishing progress at a rate that has already rendered the initial methodologies introduced fifteen years ago obsolete. DNA extraction now can be quickly and efficiently performed by various kinds of commercially available kits. The advent of PCR has enabled the use of relatively crude and minute DNA as amplification templates while many kinds of new detection methods for analyzing the amplified products have also been developed. Although many minisatellites such as MCT118, YNZ22, COL2A1, and ApoB were highlighted at the beginning of 1980s, none of these loci, with the exception of MCT118, have proved useful for forensic DNA application due to their low amplification efficiency. On the other hand, STR loci containing four base pair repeat sequences have been used routinely for human identification since the mid-1990s. In the near future, the highly efficient STR should be selected as a consensus core marker in Japan. STR systems located on the Y chromosome are widely used in forensic science for the identification of male individuals. These systems have a special significance in forensic science cases where mixtures of male and female DNA are analyzed, as happens in cases of rape or other sexual crimes. The characteristics of high copy number, maternal inheritance, and high degree of sequence variability make mtDNA a powerful tool for forensic identification. Most of the variations in mtDNA among individuals are found within the displacement loop (D-loop). In all population groups, mtDNA sequences can be useful for discriminating among unrelated individuals. Now it is necessary to get as much as possible individual genetic information as quickly as possible in order to enable individual identification. We will create a new era in which forensic identification can be performed using microarray technology.

Blotting, Southern↗

A knob-associated tandem repeat in maize capable of forming fold-back DNA segments: are chromosome knobs megatransposons?

A class of tandemly repeated DNA sequences (TR-1) of 350-bp unit length was isolated from the knob DNA of chromosome 9 of Zea mays L. Comparative fluorescence in situ hybridization revealed that TR-1 elements are also present in cytologically detectable knobs on other maize chromosomes in different proportions relative to the previously described 180-bp repeats. At least one knob on chromosome 4 is composed predominantly of the TR-1 repeat. In addition, several small clusters of the TR-1 and 180-bp repeats have been found in different chromosomes, some not located in obvious knob heterochromatin. Variation in restriction fragment fingerprints and copy number of the TR-1 elements was found among maize lines and among maize chromosomes. TR-1 tandem arrays up to 70 kilobases in length can be interspersed with stretches of 180-bp tandem repeat arrays. DNA sequence analysis and restriction mapping of one particular stretch of tandemly arranged TR-1 units indicate that these elements may be organized in the form of fold-back DNA segments. The TR-1 repeat shares two short segments of homology with the 180-bp repeat. The longest of these segments (31 bp; 64% identity) corresponds to the conserved region among 180-bp repeats. The polymorphism and complex structure of knob DNA suggest that, similar to the fold-back DNA-containing giant transposons in Drosophila, maize knob DNA may have some properties of transposable elements.

Base Sequence↗

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↗

Chloroplast DNA variation and reticulate evolution in sexual and apomictic sections of dandelions.

Sequencing of the trnL-trnF intergenic spacer in chloroplast DNA (cpDNA) from 237 sexual and apomictic species of dandelions (genus Taraxacum) from Europe, Asia and arctic North America revealed 46 haplotypes, which differed mainly by a variable number of polymorphic tRNA pseudogenes next to the trnF gene. The haplotypes could be divided into 20 cpDNA lineages, but independent duplications and deletions of the pseudogene copies made it difficult to further reconstruct the phylogeny. Intraspecific cpDNA variation was found in the primitive sexual T. serotinum. However, in contrast to a recent study, no cpDNA variation was detected within 12 apomictic species representing a variety of haplotypes. The cpDNA haplotype may therefore help to define these critical apomicts. On the other hand, the genetic variation may easily be overestimated, if the clones are not correctly identified, because some morphologically similar microspecies carried very different haplotypes. In all, 36 sections of the genus were sampled. Four primitive, mainly sexual, sections only displayed a group of ancient haplotypes, whereas morphologically more advanced sections often exhibited many different haplotypes from up to seven cpDNA lineages. In the latter cases, the lineages were rarely unique to a certain section. For example, the two most widespread haplotypes, belonging to different lineages, were found together in nine sections. This suggests that significant gene flow has occurred among the advanced sections, although sexual reproduction is not currently known in several of them. The result is consistent with the reticulate distribution of morphological characters among the sections.

Asia↗

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↗

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↗

Quantification of total genomic DNA and selected repetitive sequences reveals concurrent changes in different DNA families in indica and japonica rice.

This paper describes a fluorescence in situ hybridization (FISH) analysis of three different repetitive sequence families, which were mapped to mitotic metaphase chromosomes and extended DNA fibers (EDFs) of the two subspecies of rice (OrYza sativa), indica and japonica (2n = 2x = 24). The repeat families studied were (1) the tandem repeat sequence A (TrsA), a functionally non-significant repeat; (2) the [TTTA-GGG]n telomere sequence, a non-transcribed, tandemly repeated but functionally significant repeat; and (3) the 5S ribosomal RNA (5S rDNA). FISH of the TrsA repeat to metaphase chromosomes of indica and japonica cultivars revealed clear signals at the distal ends of twelve and four chromosomes, respectively. As shown in a previous report, the 17S ribosomal RNA genes (17S rDNA) are located at the nucleolus organizers (NORs) on chromosomes 9 and 10 of the indica cultivar. However, the japonica rice lacked the rDNA signals on chromosome 10. The size of the 5S rDNA repeat block, which was mapped on the chromosome 11 of both cultivars, was 1.22 times larger in the indica than in the japonica genome. The telomeric repeat arrays at the distal ends of all chromosome arms were on average three times longer in the indica genome than in the japonica genome. Flow cytometric measurements revealed that the nuclear DNA content of indica rice is 9.7% higher than that of japonica rice. Our data suggest that different repetitive sequence families contribute significantly to the variation in genome size between indica and japonica rice, though to different extents. The increase or decrease in the copy number of several repetitive sequences examined here may indicate the existence of a directed change in genome size in rice. Possible reasons for this phenomenon of concurrent evolution of various repeat families are discussed.

Blotting, Southern↗

Quantitation of HCV RNA using real-time PCR and fluorimetry.

Real-time PCR technology may provide an accurate and sensitive method to quantify hepatitis C virus (HCV) RNA. So far, studies have been carried out using the Taqman technology with the ABI Prism 7700 sequence detector. An alternative and simple real-time PCR assay is described with no probe requirement, based on the SYBR Green I dye and LightCycler fluorimeter. Amplicon synthesis was monitored continuously by SYBR Green I dye binding to double stranded DNA during PCR of the 5' HCV non-coding (NC) region. Specificity was verified by amplicon melting temperatures. An external standard curve was constructed with serial 10 fold dilutions of a modified synthetic HCV 5' NC RNA. A wide range linear relationship (up to 3.7x10(9) copies/ml) was observed between number of PCR cycle needed to detect a fluorescent signal and number of RNA copy. Intra- and inter-assay coefficients of variation were 0.7 to 2.1 and 3.7% respectively, indicating good reproducibility of the method. Thirty-three HCV positive sera of different genotypes were quantified by this method and gave similar but more sensitive results compared to the branched DNA (bDNA) technology.

Fluorometry↗

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↗

Intratumoral regional variations in copy number of the chromosomal part revealed by microdissection and combined ploidy and comparative genomic hybridization analyses in esophageal squamous cell carcinoma.

Intratumoral regional variations in the copy number of chromosomal material were analyzed to demonstrate the time sequence of chromosomal changes in progression of individual squamous cell carcinoma of the esophagus. We applied combined DNA ploidy and comparative genomic hybridization (CGH) analyses to multiple DNA samples extracted from microdissected, formalin-fixed, paraffin-embedded tissues, and amplified and labeled according to degenerate oligonucleotide-primed polymerase chain reaction. We examined two cases: one with a deep invasive tumor and the other with a superficial spreading tumor. We found that each sample had unique aberrations in addition to the ones common to all or some of the samples in a tumor. Based on previous studies (Okada et al., Cancer Genet Cytogenet 2000;118:99-107), we classified significant shifts of the green to red (G/R) ratio into small and large ones, which were within and beyond the range of 0.65 to 1.35, respectively. Most of the large-shift aberrations were found to be common to all or some of the samples in each case. These were thought to represent earlier events in the DNA-diploid stage, while small shifts may possibly reflect one-copy changes after tetraploidization or chromosomal instability. Based on the breakpoints and on the absolute copy numbers of altered chromosomal parts inferred from DNA ploidy and the shift size of the G/R ratio, we reconstructed the sequence of accumulation and divergence of chromosomal alterations as a dendrogram in each case. This method of temporal analysis may enable us to extract important early events from numerous aberrations screened by CGH in individual tumors.

Anatomy, Regional↗

Use of PCR-RFLP assays to detect genetic variation at single-copy nuclear loci in striped bass (Morone saxatilis).

We developed three assays that detect genetic variation at single-copy nuclear loci in striped bass (Morone saxatilis). Because these assays are based on restriction enzyme digests of DNA amplified by the polymerase chain reaction (PCR-RFLP), they are easy to perform on large numbers of samples. Breeding trials demonstrated that the alleles identified in each of the three assays are inherited in a Mendelian fashion as codominant alleles at single-copy loci. To demonstrate the utility of these PCR-RFLP assays, we compared the genetic composition of striped bass populations from the Congaree River in South Carolina and from the Choptank River in Maryland. Allele frequencies were significantly different at the SB14 locus, suggesting that the two populations may be genetically distinct. Furthermore, during the development of the PCR-RFLP assays, we demonstrated that the GT(n) microsatellite-associated DNA regions (MSA regions) contained RFLPs at a frequency 9-fold higher than that observed for randomly chosen segments of DNA. If MSA regions proved to be variable in other organisms as well, they could provide a valuable source of intraspecific variation.

Animals↗