麂亚科动物钾离子通道基因片段及其内含子 序列的克隆与进化分析

运用PCR扩增、T-A克隆、测序等技术,获得黑麂,小麂,赤麂和毛冠鹿4种麂亚科动物基因组DNA的钾离子通道部分序列。序列分析表明:麂属动物之间的外显子区域序列差异为0.90%～1.44%,毛冠鹿与麂属动物之间的差异为0.90%～1.26%,可见这一段序列的同源性较高。而内含子部分序列差异在麂属动物之间的差异为0～1.22%,毛冠鹿与麂属动物的差异为1.83%左右。由NJ法和最大简约法（Mp法）构建的进化树表明黑麂与赤麂亲缘关系较近,小麂与他们同为一属但关系较远,而毛冠鹿与它们之间的分化程度已达到属间水平。研究表明基因组的内含子序列能够较真实反映近缘动物之间的关系,是进行分子进化比较分析的较理想标记。 Abstract: In this study, partial fragments of potassium ion channel gene were amplified using the genomic DNA of muntjak, reevesi, crinifrons, and Elaphodus cephalophus. The PCR products were ligated to the plasmid of pMD18-T Vector by the method of direct T-A cloning. The positive clones were identified by colony PCR. The sequences of the recombinant clones were determined using M13-47/RV-M universal primers and aligned by the software CLUSTALW. The nucleotide divergences of exon were 0.90%-1.44% among three species of Muntiacus, 0.90%-1.26% between E. cephalophus and each of Muntiacus deer. In the nucleotide of intron there is 0%-1.22% difference among these muntjac deers, and the divergene reached about 1.83% between E. cephalophus and the three species of Muntiacus. Using the software of MEGA to analyse molecular phylogeny, Phylogenetic trees were constructed with neighbor-joining method and maximum parsimony method. The result showed Muntiacus, crinifrons is most closely related to muntjak, with reevesi as their sister species. E. cephalophus is in the other genus.     

四甲基氯化铵在PCR扩增小麦基因中的关键作用

利用高简并性引物,用PCR法从小麦DNA或cDNA中合成小麦几丁质酶基因、葡 聚糖酶基因和苯丙氨酸解氨酶基因片段。在PCR反应中添加四甲基氯化铵(TMACl)是合成这些特异基因片段的关键。合成的PCR片段都经末端补齐和磷酸化后用于克隆。核酸序列分析证实,这些PCR产物分别与用于设计PCR引物的基因具有高度的同源性。 Abstract:In the presence of tetramethy1 ammonium chloride(TMAC1),a chitinase gene sequence,a phenylalanine ammonia-lyase gene sequence and a glucanase cDNA sequence of wheat were amplified with highly degenerate primers by PCR.The inclusion of TMAC1 in the PCR reactions was essential for successful amplification of the desired sequences from genomic DNA or cDNA in wheat.The ends of the PCR fragments were made flush and phosphorylated prior to cloning.Sequence analyses of the above PCR fragments confirmed their identities,showing high sequence similarities to the genes used for the design of PCR primers.     

SEN病毒D亚型中国分离株基因克隆及序列分析

According to the published nucleotide sequence of SEN virus genome,specific primers were designed and synthesizedFrom the serum of a Chinese patient with non-A-E hepatitis,two long fragments(totally 3175bp) spanning the complete coding region of SENV-D variant gene were amplified by semi-nested PCRThe amplified fragments were cloned and sequencedThe nucleotide sequence homology of this Chinese strain with SENV-D(AX025730),SENV-D(AB059352) and TTV(AB028668) were 90%,88% and 91% respectivelyThe protein encoded by ORF1 has two putative conserved sequence motifs relating to the replication of the virus and,besides,a conserved ATP/GTP-binding motif and several highly conserved protein kinase phosphorylation sites     

Preliminary study on the detection of the SARS-CoV specific target cDNA fragments by multiplex PCR

The multiplex polymerase chain reaction (PCR) technique was applied to detect the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) specific target cDNA fragments in the present study. The target cDNA fragments of SARS-CoV were synthesized artificially according to the genome sequence of SARS-CoV in GenBank submitted by The Chinese University of Hong Kong, and were used as simulated positive samples. Five primers recommended by World Health Organization (WHO) were used to amplify the fragments by single PCR and multiplex PCR. Three target cDNA fragments (121, 182 and 302 bp), as well as the three different combinations of any two of these fragments, were amplified by single PCR. The combination of these three fragments was amplified by multiplex PCR. The re~sults indicated that the multiplex PCR technique could be applied to detect the SARS-CoV specific target cDNA fragments successfully.     

Structural and Expressional Variations of the Mitochondrial Genome Conferring the Wild Abortive Type of Cytoplasmic Male Sterility in Rice

The so-called ＂wild abortive＂ （WA） type of cytoplasmic male sterility （CMS） derived from a wild rice species Oryza rufipogon has been extensively used for hybrid rice breeding. However, extensive analysis of the structure of the related mitochondrial genome has not been reported, and the CMS-associated gene（s） remain unknown. In this study, we exploited a mitochondrial genome-wide strategy to examine the structural and expressional variations in the mitochondrial genome conferring the CMS. The entire mitochondriai genomes of a CMS-WA line and two normal fertile rice lines were amplified by Long-polymerase chain reaction into tilling fragments of up to 15.2 kb. Restriction and DNA blotting analyses of these fragments revealed that structural variations occurred in several regions in the WA mitochondrial genome, as compared to those of the fertile lines. All of the amplified fragments covering the entire mitochondrial genome were used as RNA blot probes to examine the mitochondriai expression profile among the CMS-WA and fertile lines. As a result, only two mRNAs were found to be differentially expressed between the CMS-WA and the fertile lines, which were detected by a probe containing the nad5 and orf153 genes and the other having the ribosomal protein gene rpl5, respectively. These mRNAs are proposed to be the candidates for further identification and functional studies of the CMS gene.     

Amplification, analysis and chromosome mapping of novel homeobox-containing and homeobox-flanking sequences in rice

Homeobox genes, widely distributed among animal and plant kingdoms, play an important role in developmental process. Several homeobox conserved fragments were amplified by PCR and the flanking regions were also obtained by an LM-PCR procedure. Sequencing and Southern analysis showed that they belong to a homeobox gene family of rice. Six homeobox-containing fragments were mapped on the molecular linkage map of rice. They were located on chromosomes 3, 4 and 7 respectively. It is noteworthy that there are 4 homeobox fragments located on rice chromosome 3 and the result is also consistent with the comparative genomics between rice and maize.     

Phylogeny of Chinese Allium (Liliaceae) using PCR-RFLP analysis

Eighteen representative species were selected from all the nine sections of Chinese Allium on the basis of the classification of morphology and cytotaxonomy. The trnK and rpL16 gene fragments of chloroplast DNA were amplified from 18 species by PCR method. The two cpDNA fragments were digested by 26 restriction enzymes, and 303 polymorphic restriction sites were found, of which 163 were informative. The restriction site data were analyzed with PAUP (version 3.1.1) and MEGA (version 1.01) as well as PHYLIP. As a result, the genus Allium could be classified into six subgenera. The recognition of Sect. Anguinum in the Flora of China is reasonable, Sect. Rhizirideum, Sect. Haplostemon and Sect. Cepa are not monophyletic. The infrageneric system of this genus was also discussed.     

A strategy for searching antigenic regions in the SARS-CoV spike protein

In the face of the worldwide threat of severe acute respiratory syndrome (SARS) to human life, some of the most urgent challenges are to develop fast and accurate analytical methods for early diagnosis of this disease as well as to create a safe anti-viral vaccine for prevention. To these ends, we investigated the antigenicity of the spike protein (S protein), a major structural protein in the SARS-coronavirus (SARS-CoV). Based upon the theoretical analysis for hydrophobicity of the S protein, 18 peptides were synthesized. Using Enzyme-Linked Immunosorbent Assay (ELISA),these peptides were screened in the sera from SARS patients. According to these results, two fragments of the S gene were amplified by PCR and cloned into pET-32a. Both S fragments were expressed in the BL-21 strain and further purified with an affinity chromatography. These recombinant S fragments were confirmed to have positive cross-reactions with SARS sera, either by Western blot or by ELISA. Our results demonstrated that the potenti     

Structural analysis of DMD gene and its clinical application in Chinese.I.Bgl Ⅱ exon—containing fragment,RFLP and carrier detection

This article is one of the serial studies on the characteristics of the molecular structure for dystrophin gene in Chinese.By using the entire dystrophin cDNA(14kb) as a probe,the number and RFLPs of Bgl Ⅱ exon-containing fragments of the dystrophin gene were analysed.Four new Bgl Ⅱ fragments were found,two of them(3.7 and 6.2 kb) detected by comparing the hybridization patterns with cDNA1-2a,1a and 2a,one(9.3 kb) from the hybridization pattern with cDNA 9 by lengthening migrating distance of DNA fragments in electrophoresis,and another and (4.0 kb) by comparing the patterns with cDNA 11-14, 11a,11b,aac-12a and 14.The results indicated that the number of Bgl Ⅱ exon-containing fragments should be 59 rather than 55 reported previously,which laid the foundation of the Bgl Ⅱ partial restriction map for dystrophin gene.Three of the four RFLPs found in Caucacian appear in the hybridization patterns of three subclones,i.e. cDNA 2b-3,cDNA 4-5,and cDNA 5b-7.The values of expected heterozygote frequency(EHF) were 0.33,0.33 and 0.40,and the observed heterozygote frequency(OHF) were 0.40,0.40 and 0.48 respectively.Meanwhile,two new rare allelic fragments(15kb) were found in RFLPs from Bgl Ⅱ/2b-3 and Bgl Ⅱ/4-5a patterns respectively.These Bgl Ⅱ RFLPs and four XbaI RFLPs documented in our laboratory have been used to detect the carrier in 7 DMD families and 1 BMD family.Of the 69 individuals from the 8 families,11 females were diagnosed as the carriers with DMD mutation,4 females as the doubtful carriers,12 females were defined as normal genotype and 2 females as probably normal.The results suggest that the carrier testing method based on dosage intensity analysis and genotype analysis by using dystrophin cDNA as a probe will be more sensitive and accurate.     

山羊β乳球蛋白基因的克隆及其在转基因小鼠乳汁中的高效表达 Goat β-lactoglobulin Gene Cloning and High Expression in the Mammary Gland of Transgenic Mice

通过长距离PCR从山羊基因组DNA分两段扩增山羊β乳球蛋白（β-lactoglobulin,BLG）基因,扩增出的两个片段分别克隆到T载体上,利用BLG基因序列自身存在的NarI单酶切位点进行拼接,获得了全长为7.2kb的山羊BLG基因克隆,并构建了它的真核表达载体,经酶切鉴定和序列分析证实了克隆的正确性。用线性化的BLG基因显微注射小鼠受精卵以建立转基因鼠,经PCR和Southern印迹分析证实获得了6只首建者（Founder）转基因小鼠（3♀,3♂）,在泌乳期采集两只F0代转基因雌鼠乳汁并用ELISA测定山羊β乳球蛋白的含量,其表达水平分别为23.49 mg/mL和2.19 mg/mL。 Abstract:To clone goat β-lactoglobulin (BLG) gene,two fragments were amplified from goat genomic DNA by LD-PCR method.The fragments were inserted in T-vectors before being spliced into the whole 7.2 kb BLG gene at a single restriction enzyme site of NarI.Consequently,the eukaryotic expression vector was constructed.All the clones were proved to be correct by restriction enzyme cutting and sequencing analysis.Six Founders (3♀,3♂) of goat BLG transgenic mice were obtained by microinjection and BLG genes integration were confirmed by both PCR and Southern blot analyses.The milk was collected from two lactating female transgenic mice and goat BLG protein contents were measured with ELISA.The results showed that goat BLG protein in milk of the two mice were 23.49 mg/mL and 2.19 mg/mL,respectively.     

A rapid sex-identification test for the forest musk deer (Moschus berezovskii) based on the ZFX/ZFY gene

We describe a rapid sex-identification method for the forest musk deer (Moschus berezovskii) using PCR based on zinc-finger protein-encoding genes (ZFX/ZFY) located on the X and Y chromosomes. Fragments of the ZFX and ZFY genes were amplified and sequenced. The ZFX and ZFY fragments were identical in length and 94% similar in nucleotide sequence. Specific primers for forest musk deer sex identification were designed on the basis of sequence differences between ZFX and ZFY. All the primers were multiplexed in single-tube PCR. Both male and female forest musk deer showed amplification bands of 447 bp and 212 bp separated in agarose gels. A sex-specific 278-bp band was amplified only from males. These results show that testing by PCR for the presence of the 278-bp sequence is a rapid and reliable method for sex identification.     

The sex-determining zinc finger sequences in XY females of Akodon azarae (Rodentia, Cricetidae)

Wild populations of Akodon azarae comprise females with a karyotype indistinguishable from that of males. These individuals were formerly assumed to be Xx, the x being an X chromosome with a deletion of most of its long arm. By using a DNA probe derived from the testis-determining region of the human Y chromosome (comprising a candidate gene for the testis-determining factor, Y-linked zinc finger [ZFY]), we demonstrate that A. azarae gonosomally variant females are XY and not Xx. The ZFY sequences in A. azarae are amplified and located in two different families of EcoRI fragments derived from Y-chromosome DNA. No rearrangement or change in the state of methylation of ZFY or ZFX (X-linked zinc finger) sequences were found in XY females. We propose that sex reversal in A. azarae may be mediated by a gene or genes other than ZFX or ZFY.     

Polymorphic karyotypes and sex chromosomes in the tufted deer (Elaphodus cephalophus): cytogenetic studies and analyses of sex chromosome-linked genes

Different diploid chromosome numbers have been reported for the tufted deer Elaphodus cephalophus (female, 2n = 46/47; male, 2n = 47/48) in earlier reports. In the present study, chromosomal analysis of seven tufted deer (5 male symbol, 2 female symbol) revealed that the karyotype of these animals contains 48 chromosomes, including a pair of large heteromorphic chromosomes in the male. C-banding revealed these chromosomes to be very rich in constitutive heterochromatin. Chromosome banding and PCR of sex chromosome-linked genes (SRY, ZFX, ZFY) performed on DOP-PCR products of single microdissected X and Y chromosomes confirmed that the large telocentric chromosome without secondary constriction is the X chromosome whereas the subtelocentric chromosome is the Y. The increased size of both, the X and Y chromosome, appears to be at least partially attributable to the presence of substantial amounts of heterochromatin.     

Evolution of the X-linked Zinc Finger Gene and the Y-linked Zinc Finger Gene in Primates

We have sequenced the partial exon of the zinc finger genes (ZFX and ZFY) in 5 hominoids, 2 Old World monkeys, 1 New World monkey, and 1 prosimian. Among these primate species, the percentage similarities of the nucleotide sequence of the ZFX gene were 96-100% and 91.2-99.7% for the ZFY gene. Of 397 sites in the ZFX and ZFY gene sequences, 20 for ZFX gene and 42 for ZFY gene were found to be variable. Substitution causes 1 amino acid change in ZFX, and 5 in ZFY, among 132 amino acids. The numbers of synonymous substitutions per site (Ks) between human and the chimpanzee, gorilla and orangutan for ZFY gene were 0.026, 0.033, and 0.085, respectively. In contrast, the Ks value between human and hominoid primates for the ZFX gene was 0.008 for each comparison. Comparison of the ZFX and ZFY genes revealed that the synonymous substitution levels were higher in hominoids than in other primates. The rates of synonymous substitution per site per year were higher in the ZFY exon than in the SRY exon, and higher in the ZFY exon than in the ZFY intron, in hominoid primates.     

Sex determination in spotted hyena (Crocuta crocuta ) by restriction fragment length polymorphism of amplified ZFX/ZFY loci

We describe a quick and efficient method of determining the sex of DNA samples in the hyena. By choosing primers from sequences that are conserved between the human and bovine ZFY and ZFX genes, we amplified a 448 bp fragment from 1 male and 2 female hyenas. Using comparative sequencing, single base pair polymorphisms between the amplified ZFY and ZFX were established. Restriction fragment length polymorphism (RFLP) analysis with PstI and TaqI confirmed the sequence data and yielded specific banding patterns between the 2 sexes in the hyena.     

No evidence of mutations in four candidate genes for male sex determination/differentiation in sex-reversed XY females with campomelic dysplasia.

Campomelic dysplasia (Cd) occurs combined with sex reversal resulting in XY females. The recent identification of candidate genes for sex determination/differentiation and of a sex determining region on the human Y chromosome prompted the authors to study these genes for mutations in patients with Cd and sex reversal. In a total of five cases, no evidence for a mutation in the genes SRY, ZFY, ZFX, MEA and some anonymous Y-linked sequences was found. In addition to Southern analysis, gene expression of ZFY, ZFX and MEA was found to be normal as well. It is concluded that sex reversal in this condition is due to mutation in a so far unidentified gene which may act secondary to the testis-determining factor (TDF).     

Identification of sex in Cetaceans by multiplexing with three ZFX and ZFY specific primers

We sequenced 540 nucleotides of the last exon in the ZFY/ZFX gene in two males and two females for eight cetacean species; four odontocetes (toothed whales) and four mysticetes (baleen whales). Based upon the obtained nucleotide sequences, we designed two sets of oligonucleotide primers for specific amplification of the ZFX and the ZFY sequence in odontocetes and mysticetes, respectively. Each primer set consisted of three oligonucleotides; one forward-orientated primer, which anneals to the ZFY as well as the ZFX sequence, and two reverse-orientated primers that anneal to either the ZFX or the ZFY sequence. The resulting two amplification products (specific for the ZFY and ZFX sequences) can be distinguished by gel-electrophoresis through 2% NuSieve™. The accuracy of the technique was tested by determination of gender in 214 individuals of known sex. Finally we applied the technique to determine the sex of 3570 cetacean specimens; 2284 humpback whales, 315 fin whales, 37 blue whales, 7 minke whales, as well as 592 belugas, 335 narwhals and 25 harbour porpoises.     

Cloning and comparative analysis of the bovine, porcine, and equine sex chromosome genes ZFX and ZFY.

A growing body of evidence suggests the involvement of sex chromosome genes in mammalian development. We report the cloning and characterization of the complete coding regions of the bovine Y chromosome ZFY and X chromosome ZFX genes, and partial coding regions of porcine and equine ZFX and ZFY genes. Bovine ZFY and ZFX are highly similar to each other and to ZFX and ZFY from other species. While bovine and human ZFY proteins are both 801 amino acids long, bovine ZFX is 5 amino acids shorter than human ZFX. Like in humans, both bovine ZFY and ZFX contain 13 zinc finger motifs and belong to the Krueppel family of C2H2-type zinc finger proteins. The internal exon-intron organization of the bovine, porcine and equine ZFX and ZFY genes has been determined and compared. Within this region, the exon lengths and the positions of the splice sites are conserved, further suggesting a high evolutionary conservation of the ZFX and ZFY genes. Additionally, new alternatively spliced forms of human ZFX have been identified.