青海喇家遗址人类遗骸的锶同位素比值分析

喇家遗址位于青海省民和县,主体为齐家文化的遗存,距今大约3900~4300年。喇家遗址古代人类迁移活动的研究,对于探索黄河上游古代文明,推动该地区齐家文化研究,有着积极的学术意义。本文首先采用电感耦合等离子体质谱技术对喇家遗址出土的22个个体的44份牙釉质和骨骼样品进行了元素分析,根据检测结果排除了受到污染的2份骨骼样品。其次,通过热电离质谱分析方法对喇家遗址出土22个个体的42份牙釉质和骨骼样品及8个猪牙釉质样品的锶同位素比值进行了测定。检测数据表明,猪牙釉质的锶同位素比值的标准偏差远小于其他动物,可以推断猪由当地饲养的可能性是最大的,因此其牙釉质锶同位素比值可以代表当地的锶同位素水平。经过计算得到8个猪牙釉质的锶同位素比值的平均值为0.710930,将该平均值加或减2倍标准偏差可以得到喇家遗址当地的锶同位素比值范围在0.711058~0.710802之间。以此为前提可以推测,本次检测的22个个体中,有17个个体牙釉质的锶同位素比值在遗址当地的锶同位素比值范围内,可能为本地出生。还有2个个体牙釉质在当地的锶同位素比值范围上下限附近,姑且存疑;仅有3个个体牙釉质的锶同位素比值在遗址当地的锶同位素比值范围以外,表明这些个体可能在其他地区出生,去世后埋葬在遗址中。此外,数据分析还显示,本地出生人群牙釉质的锶同位素比值平均值与骨骼的锶同位素比值平均值比较接近,暗示喇家遗址当地古人类生活方式可能比较统一。     

Ancient DNA evidence supports the contribution of Di‐Qiang people to the han Chinese gene pool

Han Chinese is the largest ethnic group in the world. During its development, it gradually integrated with many neighboring populations. To uncover the origin of the Han Chinese, ancient DNA analysis was performed on the remains of 46 humans (～1700 to 1900 years ago) excavated from the Taojiazhai site in Qinghai province, northwest of China, where the Di‐Qiang populations had previously lived. In this study, eight mtDNA haplogroups (A, B, D, F, M*, M10, N9a, and Z) and one Y‐chromosome haplogroup (O3) were identified. All analyses show that the Taojiazhai population presents close genetic affinity to Tibeto‐Burman populations (descendants of Di‐Qiang populations) and Han Chinese, suggesting that the Di‐Qiang populations may have contributed to the Han Chinese genetic pool. Am J Phys Anthropol, 2011. © 2010 Wiley‐Liss, Inc.     

Monotropa uniflora: morphological and molecular assessment of mycorrhizae retrieved from sites in the sub-boreal spruce biogeoclimatic zone in central British Columbia

Plant species in the subfamily Monotropoideae are achlorophyllous and have developed a complex mode of nutrition, receiving photosynthates from neighboring trees via shared fungi. To explore the mycorrhizal associations of Monotropa uniflora in central British Columbia (B.C.), plants were sampled from three sites: a Betula-dominated site and two sites with a mixture of conifer and hardwood trees. Fifteen M. uniflora root-clusters were sampled (five per site) and the mycorrhizal diversity was assessed using morphological and molecular (PCR-RFLP analysis and DNA sequencing) methods. Both methods showed that root-clusters (often comprising several hundred mycorrhizal tips) belonging to the same plant appeared to involve fungus monocultures in the family Russulaceae. All mycorrhizae exhibited typical Russula morphology and had mantle cystidia. Two root-clusters, one each from sites 1 and 3, lacked one of the two types of cystidia present on all other root-clusters. PCR-RFLP analysis resulted in three fragment patterns for the 15 root clusters. One molecular fragment pattern included the two root-clusters displaying the single cystidium type plus an additional root-cluster with both cystidia types. DNA sequencing of a portion of the ITS2 region of the ribosomal DNA suggests that the three variants represent different species; two of the variants clustered with the hypogeous fungi Martellia and Gymnomyces. The study provides increased evidence of low diversity and high specificity in the Monotropa-fungus relationship and suggests that M. uniflora associates uniquely with fungi in the family Russulaceae in central B.C.     

Deletion analysis of the cloned replication origin region from bacteriophage M13.

A cloned 270-nucleotide fragment from the origin region of the M13 duplex replicative form DNA confers an M13-dependent replication mechanism upon the plasmid vector pBR322. This M13 insert permits M13 helper-dependent replication of the hybrid plasmid in polA cells which are unable to replicate the pBR322 replicon alone. Using in vitro techniques, we have constructed several plasmids containing deletions in the M13 DNa insert. The endpoints of these deletions have been determined by DNA sequence analysis and correlated with the transformation and replication properties of each plasmid. Characterization of these deletion plasmids allows the following conclusions. (i) The initiation site for M13 viral strand replication is required for helper-dependent propagation of the chimeric plasmid. (ii) A DNA sequence in the M13 insert, localized between 89 and 129 nucleotides from the viral strand initiation site, is necessary for efficient transformation of polA cells. A chimeric plasmid containing the viral strand initiation site, but lacking this additional 40 nucleotide M13 sequence, transforms helper-infected cells at a frequency approximately 10(4)-fold less than that of plasmids containing this additional DNA segment. (iii) The entire M13 complementary strand origin can be deleted without affecting M13-dependent transformation by the hybrid plasmids. We propose a model in which replication of one strand of duplex chimera initiates by nicking at the gene II protein nicking site in the viral strand of the M13 insert, followed by asymmetric single-strand synthesis. Initiation of the complementary strand possibly occurs within plasmid sequences.     

Roles of the helicase and primase domain of the gene 4 protein of bacteriophage T7 in accessing the primase recognition site.

The 63 kDa gene 4 protein of bacteriophage T7 provides both helicase and primase activities. The C-terminal helicase domain of the gene 4 protein is responsible for DNA-dependent NTP hydrolysis and for hexamer formation, whereas the N-terminal primase domain contains the zinc motif that is, in part, responsible for template-directed oligoribonucleotide synthesis. In the presence of beta, gamma-methylene dTTP, the protein forms a hexamer that surrounds and binds tightly to single-stranded DNA and consequently is unable to translocate to primase recognition sites, 5'-GTC-3', or to dissociate from the molecule to which it is bound. Nonetheless, in the presence of beta,gamma-methylene dTTP, it catalyzes the synthesis of pppAC dimers at primase sites on M13 DNA. When bound to single-stranded DNA in the presence of beta,gamma-methylene dTTP, the primase can function at recognition sites on the same molecule to which it is bound provided that a sufficient distance exists between the recognition site and the site to which it is bound. Furthermore, the primase bound to one DNA strand can function at a primase site located on a second DNA strand. The results indicate that the primase domain resides on the outside of the hexameric ring, a location that enables it to access sites distal to its site of binding.     

Mitochondrial DNA analysis of Jomon skeletons from the Funadomari site, Hokkaido, and its implication for the origins of Native American

Ancient DNA recovered from 16 Jomon skeletons excavated from Funadomari site, Hokkaido, Japan was analyzed to elucidate the genealogy of the early settlers of the Japanese archipelago. Both the control and coding regions of their mitochondrial DNA were analyzed in detail, and we could securely assign 14 mtDNAs to relevant haplogroups. Haplogroups D1a, M7a, and N9b were observed in these individuals, and N9b was by far the most predominant. The fact that haplogroups N9b and M7a were observed in Hokkaido Jomons bore out the hypothesis that these haplogroups are the (pre-) Jomon contribution to the modern Japanese mtDNA pool. Moreover, the fact that Hokkaido Jomons shared haplogroup D1 with Native Americans validates the hypothesized genetic affinity of the Jomon people to Native Americans, providing direct evidence for the genetic relationships between these populations. However, probably due to the small sample size or close consanguinity among the members of the site, the frequencies of the haplogroups in Funadomari skeletons were quite different from any modern populations, including Hokkaido Ainu, who have been regarded as the direct descendant of the Hokkaido Jomon people. It appears that the genetic study of ancient populations in northern part of Japan brings important information to the understanding of human migration in northeast Asia and America.     

Identification of the nucleotide sequences specific for the 5'-flanking regions of the genes regulated by glucocorticoids by a computer analysis method

Nucleotide sequences of 5'-flanking regions of 11 glucocorticoid-regulated genes and 14 genes non-regulated by these hormones were studied using context computer analysis. Consensus TGTTCT, previously found in DNA fragments protected by glucocorticoid-receptor complexes from DNAase I digestion, was shown to be nonspecific for glucocorticoid-regulated genes. However, the analysis of sequences flanking the TGTTCT consensus has revealed that only glucocorticoid-regulated genes contain four regularly distributed cytosine residues, one of them belonging to TGTTCT consensus. Three of cytosine residues are separated by 8-10 bp, which provides their close neighbourhood at one side of DNA double helix; the fourth extreme cytosine residue is located 6 bp from the nearest one and therefore, the complementary guanine residue is adjacent to the consensus in DNA helix. It is suggested that the consensus itself and two flanking cytosine and one guanine residues form a specific site for the interaction with glucocorticoid-receptor complex.     

Cellular protein interactions with herpes simplex virus type 1 oriS.

The herpes simplex virus type 1 (HSV-1) origin of DNA replication, oriS, contains an AT-rich region and three highly homologous sequences, sites I, II, and III, identified as binding sites for the HSV-1 origin-binding protein (OBP). In the present study, interactions between specific oriS DNA sequences and proteins in uninfected cell extracts were characterized. The formation of one predominant protein-DNA complex, M, was demonstrated in gel shift assays following incubation of uninfected cell extracts with site I DNA. The cellular protein(s) that comprises complex M has been designated origin factor I (OF-I). The OF-I binding site was shown to partially overlap the OBP binding site within site I. Complexes with mobilities indistinguishable from that of complex M also formed with site II and III DNAs in gel shift assays. oriS-containing plasmid DNA mutated in the OF-I binding site exhibited reduced replication efficiency in transient assays, demonstrating a role for this site in oriS function. The OF-I binding site is highly homologous to binding sites for the cellular CCAAT DNA-binding proteins. The binding site for the CCAAT protein CP2 was found to compete for OF-I binding to site I DNA. These studies support a model involving the participation of cellular proteins in the initiation of HSV-1 DNA synthesis at oriS.     

A comparison of DNA extraction procedures for the detection of Mycobacterium ulcerans, the causative agent of Buruli ulcer, in clinical and environmental specimens

Mycobacterium ulcerans is the causative agent of Buruli ulcer, the third most common mycobacterial disease in humans after tuberculosis and leprosy. Although the disease is associated with aquatic ecosystems, cultivation of the bacillus from the environment is difficult to achieve. Therefore, at the moment, research is based on the detection by PCR of the insertion sequence IS2404 present in M. ulcerans and some closely related mycobacteria. In the present study, we compared four DNA extraction methods for detection of M. ulcerans DNA, namely the one tube cell lysis and DNA extraction procedure (OT), the FastPrep procedure (FP), the modified Boom procedure (MB), and the Maxwell 16 Procedure (M16). The methods were performed on serial dilutions of M. ulcerans, followed by PCR analysis with different PCR targets in M. ulcerans to determine the detection limit (DL) of each method. The purity of the extracted DNA and the time and effort needed were compared as well. All methods were performed on environmental specimens and the two best methods (MB and M16) were tested on clinical specimens for detection of M. ulcerans DNA. When comparing the DLs of the DNA extraction methods, the MB and M16 had a significantly lower DL than the OT and FP. For the different PCR targets, IS2404 showed a significantly lower DL than mlsA, MIRU1, MIRU5 and VNTR6. The FP and M16 were considerably faster than the MB and OT, while the purity of the DNA extracted with the MB was significantly higher than the DNA extracted with the other methods. The MB performed best on the environmental and clinical specimens. This comparative study shows that the modified Boom procedure, although lengthy, provides a better method of DNA extraction than the other methods tested for detection and identification of M. ulcerans in both clinical and environmental specimens.     

Segregation of a M404V mutation of the p62/sequestosome 1 (p62/SQSTM1) gene with polyostotic Paget's disease of bone in an Italian family

Mutations of the p62/Sequestosome 1 gene (p62/SQSTM1) account for both sporadic and familial forms of Paget's disease of bone (PDB). We originally described a methionine-->valine substitution at codon 404 (M404V) of exon 8, in the ubiquitin protein-binding domain of p62/SQSTM1 gene in an Italian PDB patient. The collection of data from the patient's pedigree provided evidence for a familial form of PDB. Extension of the genetic analysis to other relatives in this family demonstrated segregation of the M404V mutation with the polyostotic PDB phenotype and provided the identification of six asymptomatic gene carriers. DNA for mutational analysis of the exon 8 coding sequence was obtained from 22 subjects, 4 PDB patients and 18 clinically unaffected members. Of the five clinically ascertained affected members of the family, four possessed the M404V mutation and exhibited the polyostotic form of PDB, except one patient with a single X-ray-assessed skeletal localization and one with a polyostotic disease who had died several years before the DNA analysis. By both reconstitution and mutational analysis of the pedigree, six unaffected subjects were shown to bear the M404V mutation, representing potential asymptomatic gene carriers whose circulating levels of alkaline phosphatase were recently assessed as still within the normal range. Taken together, these results support a genotype-phenotype correlation between the M404V mutation in the p62/SQSTM1 gene and a polyostotic form of PDB in this family. The high penetrance of the PDB trait in this family together with the study of the asymptomatic gene carriers will allow us to confirm the proposed genotype-phenotype correlation and to evaluate the potential use of mutational analysis of the p62/SQSTM1 gene in the early detection of relatives at risk for PDB.     

矩阵设计检测金针菇转化子外源DNA插入位点

插入位点分析对于金针菇功能基因组学的研究极为重要,分析方法常用反向PCR、热不对称交错PCR、Tail-PCR、染色体步移等,存在操作复杂、消耗时间长、特异性较差、效率低等缺点。近年来开始应用基因组重测序的方法,对转化子逐一测序与分析,工作量较大、费用较高。本研究应用矩阵设计,把多个转化子的DNA混合构成样品池,重测序后分析插入位点,M个样品池的测序数据可分析M×(M+1)/2个转化子的插入位点。应用矩阵设计构建6个样品池检测21个转化子,获得21个插入位点,表明这种方法可行、适合大样本分析,如突变体库。     

Integration of the DNA of filamentous bacteriophage Cflt into the chromosomal DNA of its host.

It was demonstrated for the first time that filamentous bacteriophage Cflt, which contains single-stranded DNA, can incorporate its genome into that of its host. Evidence in support of the incorporation was obtained from a Southern blot hybridization analysis of DNA isolated from Cflt-lysogenized cells. DNAs from different Cflt-lysogenized cells were purified, and the integration patterns were compared. Because all integration patterns were identical and only one fragment in Cflt replicative-form DNA was missing, it appears that the integration was site specific. Only one complement of viral DNA was integrated per host chromosome. To determine the attachment site on the viral DNA, the physical map of EcoRI, XhoI, SstII, and BglII on Cflt DNA was constructed. Based on this physical map and a Southern blot hybridization analysis of lysogen DNA with these restriction endonucleases, we demonstrated that DNA sequences from all regions of the Cflt genome were represented in the integrated viral sequences. The attachment site on the viral genome was located at 69.2 to 73.8 min on the Cflt DNA.     

Transgene constructs in coho salmon (Oncorhynchus kisutch) are repeated in a head-to-tail fashion and can be integrated adjacent to horizontally-transmitted parasite DNA

Currently, little information is available regarding the molecular organization of integrated transgenes in genetically-engineered fish. We performed a detailed structural analysis of an inserted transgene in one strain (M77) of transgenic coho salmon (Oncorhynchus kisutch) containing a salmon growth hormone gene construct (OnMTGH1). Microinjected DNA was found to have inserted into a single site in the coho salmon genome, and was organized with four complete internal copies and two partial terminal copies of the OnMTGH1 construct. All construct copies were organized in a direct-tandem (head-to-tail) repeat fashion in strain M77 and five additional strains (one also possessed a second recombinant junction fragment). For strain M77, the junctions between the transgene insert and the insertion point within the wild-type genome were cloned from strain-specific cosmid libraries and sequenced, revealing that the transgene insertion was accompanied by a deletion of 587 bp of wild-type DNA as well as a small insertion (19 bp) of unknown DNA upstream and a 14 bp direct- tandem duplication of sequence downstream. Upstream and downstream wild-type DNA sequence contained several repetitive sequence elements based on Southern blot analysis and homology to repetitive sequences in GenBank. In the downstream flank, a pseudogene sequence was also identified which has high homology to the CA membrane protein gene from Schistosoma japonicum, a parasite closely related to Sanguinicola sp. parasites which infect salmonids. Whether the presence of an inserted transgene and the presence of potentially horizontally-transmitted DNA are indicative of a genomic region with a predisposition for insertion of foreign DNA requires further study. The information derived from this transgene structure provides information useful for comparison to other transgenic organisms and for determination of the mechanism of transgene integration in lower vertebrates.     

Acquisition of telomere repeat sequences by transfected DNA integrated at the site of a chromosome break.

Previous analysis of plasmid DNA transfected into 108 cell clones demonstrated extensive polymorphism near the integration site in one clone. This polymorphism was apparent by Southern blot analysis as diffuse bands that extended over 30 kb. In the present study, nucleotide sequence analysis of cloned DNA from the integration site revealed telomere repeat sequences at the ends of the integrated plasmid DNA. The telomere repeat sequences at one end were located at the junction between the plasmid and cell DNA. The telomere repeat sequences at the other end were located in the opposite orientation in the polymorphic region and were shown by digestion with BAL 31 to be at the end of the chromosome. Telomere repeat sequences were not found at this location in the plasmid or parent cell DNA. Although the repeat sequences may have been acquired by recombination, a more likely explanation is that they were added to the ends of the plasmid by telomerase before integration. Comparison of the cell DNA before and after integration revealed that a chromosome break had occurred at the integration site, which was shown by fluorescent in situ hybridization to be located near the telomere of chromosome 13. These results demonstrate that chromosome breakage and rearrangement can result in interstitial telomere repeat sequences within the human genome. These sequences could promote genomic instability, because short repeat sequences can be recombinational hotspots. The results also show that DNA rearrangements involving telomere repeat sequences can be associated with chromosome breaks. The introduction of telomere repeat sequences at spontaneous or ionizing radiation-induced DNA strand breaks may therefore also be a mechanism of chromosome fragmentation.     

Site-directed mutagenesis defines the catalytic aspartate in the active site of the atypical DNA: m4C methyltransferase M.NgoMXV.

M.NgoMXV is one of the few atypical DNA:m4C methyltransferases that does not possess a serine residue in its predicted active site. We previously reported a homology model of M.NgoMXV and argued that the aspartate side chain at a corresponding position, similarly to some DNA:m6 A-specific enzymes, is essential for the methyltransferase activity (Radlinska et al., 1999). Here we report the corrected amino acid sequence of M.NgoMXV and the analysis of substitution of D68 with alanine or serine, which both render the enzyme totally inactive.     

High-resolution mapping of the origin of DNA replication in the hamster dihydrofolate reductase gene domain by competitive PCR.

By the use of a highly sensitive mapping procedure allowing the identification of the start sites of DNA replication in single-copy genomic regions of untreated, exponentially growing cultured cells (M. Giacca, L. Zentilin, P. Norio, S. Diviacco, D. Dimitrova, G. Contreas, G. Biamonti, G. Perini, F. Weighardt, S. Riva, and A. Falaschi, Proc. Natl. Acad. Sci. USA 91:7119-7123, 1994), the pattern of DNA replication of the Chinese hamster dihydrofolate reductase (DHFR) gene domain was investigated. The method entails the purification of short stretches of nascent DNA issuing from DNA replication origin regions and quantification, within this sample, of the abundance of different adjacent segments by competitive PCR. Distribution of marker abundance peaks around the site from which newly synthesized DNA had emanated. The results obtained by analysis of the genomic region downstream of the DHFR single-copy gene in asynchronous cultures of hamster CHO K1 cells are consistent with the presence of a single start site for DNA replication, located approximately 17 kb downstream of the gene. This site is coincident with the one detected by other studies using different techniques in CHO cell lines containing an amplified DHFR gene domain.     

Y chromosomes of prehistoric people along the Yangtze River

The ability to extract mitochondrial and nuclear DNA from ancient remains has enabled the study of ancient DNA, a legitimate field for over 20 years now. Recently, Y chromosome genotyping has begun to be applied to ancient DNA. The Y chromosome haplogroup in East Asia has since caught the attention of molecular anthropologists, as it is one of the most ethnic-related genetic markers of the region. In this paper, the Y chromosome haplogroup of DNA from ancient East Asians was examined, in order to genetically link them to modern populations. Fifty-six human remains were sampled from five archaeological sites, primarily along the Yangtze River. Strict criteria were followed to eliminate potential contamination. Five SNPs from the Y chromosome were successfully amplified from most of the samples, with at least 62.5% of the samples belonging to the O haplogroup, similar to the frequency for modern East Asian populations. A high frequency of O1 was found in Liangzhu Culture sites around the mouth of the Yangtze River, linking this culture to modern Austronesian and Daic populations. A rare haplogroup, O3d, was found at the Daxi site in the middle reaches of the Yangtze River, indicating that the Daxi people might be the ancestors of modern Hmong-Mien populations, which show only small traces of O3d today. Noticeable genetic segregation was observed among the prehistoric cultures, demonstrating the genetic foundation of the multiple origins of the Chinese Civilization.     

The evolutionary history of the sinica-group of macaque monkeys as revealed by mtDNA restriction site analysis.

We estimated the phylogenetic relationships of mitochondrial DNA haplotypes within the sinica-group of macaques, which includes Macaca sinica, M. radiata, M. thibetana, M. assamensis, and possibly M. arctoides. Some effort was made to detect intraspecific variation by sampling individuals from different parts of the species' range or from different matrilines. In the case of M. assamensis, individuals were sampled from both subspecies (M. assamensis assamensis and M. assamensis pelops). Total genomic DNA was extracted from blood samples and cut with a battery of 16 restriction endonucleases. A total of 97 restriction sites were mapped for these enzymes in the sinica-group and M. nemestrina, which was used as an outgroup. Phylogenetic trees constructed by both the maximum parsimony method and the neighbor-joining method were highly congruent. A bootstrap analysis of the maximum parsimony tree indicated a high degree of confidence to the association of particular haplotypes within the 80% majority rule consensus tree. An exhaustive search of all possible trees also supported this topology, although one haplotype had to be eliminated from this analysis to save computer time. The results were also unaffected by weighting the character state changes in favor of site gains over site losses. The mtDNA phylogeny produced here differs from trees based on morphology and allozymes in three ways: M. sinica and M. radiata sit in two different branches of the tree; the two subspecies of M. assamensis are separated from one another; and M. arctoides consistently fell outside the rest of the sinica-group.(ABSTRACT TRUNCATED AT 250 WORDS)     

M13 vectors for selective cloning of sequences specifying initiation of DNA synthesis on single-stranded templates

M13 cloning vectors have been developed for the selection of DNA sequences capable of directing initiation of DNA synthesis on single-stranded templates. These vectors are derived from viable M13 mutants containing large deletions in the region of the complementary strand origin. The deletion mutants are defective in the conversion of viral single strands to the duplex replicative form (SS leads to RF) both in vivo and in vitro, give a reduced phage yield and form turbid plaques. A receptor site for foreign single strand initiation determinants has been introduced into the mutants by the insertion of EcoRI linker sequences at the deletion sites. Specific cloned sequences from bacteriophage G4 RF and from Co1E1 DNA restore a clear plaque type and normal phage growth. Selection of clear-plaque isolates obtained by transfection with RF from one of these vectors, M13 delta E101, carrying inserted Co1E1 HaeIII fragments resulted in the selective cloning of one specific fragment, the HaeIII-E fragment. Insertion of either the H or L strand of the HaeIII-E fragment into the M13 delta E101 viral strand gives a clear plaque phenotype, indicating the presence of initiation determinants on both the H- and L-strands of the Co1E1 HaeIII-E fragment. These cloning vectors provide a new means for the functional dissection of replication origins and for the identification of DNA sequences that determine the enzymatic mechanism of discontinuous synthesis along the length of the bacterial chromosome. The ability to assess initiation capability on the basis of plaque morphology also provides a means for rapid genetic analysis of initiation determinants.