Multiple nuclear-gene phylogenies: application to pinnipeds and comparison with a mitochondrial DNA gene phylogeny

Phylogenetic analyses of closely related species should use information from multiple, independent genes with relatively high rates of sequence evolution. To investigate species for which there are few prior sequence data for single-copy nuclear (scnDNA) genes, primers for gene amplification can be designed to highly conserved regions of exons in order to amplify both coding (exons) and noncoding (introns) sequences. We have explored this approach in a phylogenetic analysis of six species of pinnipeds that, together with terrestrial carnivore outgroups, encompass divergence times < or = 40-50 Mya. We sequenced one intron from each of the aldolase A (ALD-A), aldolase C (ALD-C), and histone H2AF genes; one exon from the major-histocompatibility-complex DQA gene; a H2AF processed pseudogene (psi H2AF); and, for comparison with the nuclear genes, the 5' portion of the mitochondrial DNA (mtDNA) control region. The pinniped psi H2AF genes were found to be of limited use because they were paralogous with the gene in the outgroup. The rate of silent substitution in scnDNA (primarily introns) was 5-10-fold lower than that for mtDNA control region I, and scnDNA sequence divergence increased linearly with time < or = 40-50 Mya. Alleles at three polymorphic scnDNA loci (ALD-A, H2AF, and DQA) in the southern elephant seal were paraphyletic with respect to the allele from the closely related northern elephant seal, while the more numerous mtDNA alleles were monophyletic. This we attribute to the consequences of a higher mutation rate rather than to a lower effective population size of mtDNA compared with scnDNA. Within the short (i.e., < 500-bp) sequences of individual scnDNA sequences, phylogenetically informative variation was insufficient to obtain robust phylogenies. However, the combined scnDNA sequences produced a well-supported phylogeny congruent with that derived from mtDNA. This analysis illustrates the high resolution of mtDNA sequences compared with a similar length of scnDNA sequence, but it also demonstrates the utility of combining information from multiple short scnDNA sequences obtained using broadly applicable primers.      

Primer Sets Developed To Amplify Conserved Genes from Filamentous Ascomycetes Are Useful in Differentiating Fusarium Species Associated with Conifers

We examined the usefulness of primer sets designed to amplify introns within conserved genes in filamentous ascomycetes to differentiate 35 isolates representing six different species of Fusarium commonly found in association with conifer seedlings. We analyzed restriction fragment length polymorphisms (RFLP) in five amplified PCR products from each Fusarium isolate. The primers used in this study were constructed on the basis of sequence information from the H3, H4, and (beta)-tubulin genes in Neurospora crassa. Primers previously developed for the intergenic transcribed spacer region of the ribosomal DNA were also used. The degree of interspecific polymorphism observed in the PCR products from the six Fusarium species allowed differentiation by a limited number of amplifications and restriction endonuclease digestions. The level of intraspecific RFLP variation in the five PCR products was low in both Fusarium proliferatum and F. avenaceum but was high in a population sample of F. oxysporum isolates. Clustering of the 35 isolates by statistical analyses gave similar dendrograms for H3, H4, and (beta)-tubulin RFLP analysis, but a dendrogram produced by intergenic transcribed spacer analysis varied in the placement of some F. oxysporum isolates.     

Haplosporidiosis in the Pacific oyster Crassostrea gigas from the French Atlantic coast

Two cases of haplosporidian infection occurred during 1993 in Pacific oysters Crassostrea gigas from the French Atlantic coast. The localization and ultrastructure of the plasmodia are described. In situ hybridization of infected tissue sections was conducted with DNA probes for oyster-infecting haplosporidians. The Haplosporidium nelsoni-specific DNA probe MSX1347 hybridized with the C. gigas parasite, and the H. costale-specific probe SSO1318 did not hybridize. Total genomic DNA was extracted from the infected tissue sections for polymerase chain reaction (PCR) amplification of the haplosporidian. PCR amplifications with H. nelsoni-specific primers and with 'universal' actin primers did not yield the expected products of 573 and 700 bp, respectively. A series of primers was designed to amplify short regions of small subunit ribosomal DNA (SSU rDNA) from most haplosporidians. The primers encompass a highly variable region of the SSU rDNA and did not amplify oyster DNA. PCR amplification of the infected C. gigas genomic DNA with these primers yielded the expected-sized product from the primer pair targeting the shortest region (94 bp). This PCR product was sequenced and it was identical to the corresponding SSU rDNA region of H. nelsoni.     

Allelic variation of ovine MHC class II DQA1 and DQA2 genes

In the present study we characterize allelic variation of polymorphic OLA-DQA1 and OLA-DQA2 genes in sheep. To achieve this, PCR primers were designed to independently amplify the second exons of OLA-DQA1 and OLA-DQA2 genes. Single strand conformation polymorphism (SSCP) gel analyses reveals that there are at least 12 distinct OLA-DQA2 sequences, 10 of which have been characterized by sequencing. Six distinct OLA-DQA1 alleles have been sequenced in sheep and we can detect at least seven DQA1 alleles, including a null allele, by SSCP analysis. The second exon of the OLA-DQA2 gene is more polymorphic than the equivalent region of the OLA-DQA1 gene. Thirty-two per cent of nucleotide and 49% of amino acid sites showed variation at the DQA2 locus, compared to 20% of nucleotide and 33% of amino acid sites for DQA1 . Phylogenetic analysis of DQA sequences from a number of species show that sheep DQA1 sequences group together and are more similar to bovine DQA1 sequences than to sheep DQA2 alleles. The majority of OLA-DQA2 sequences are on the same main branch of the phylogenetic tree as bovine DQA2 sequences. However, three sheep DQA2 sequences have a tendency to group with putative bovine DQA3 sequences rather than to other ovine DQA2 alleles. A variety of SSCP gel conditions were tried in order to develop a typing system for the OLA-DQA2 gene. We describe a set of PCR and SSCP conditions which distinguish between all known OLA-DQA2 alleles.     

Rapid and reliable identification of rice genomes by RFLP analysis of PCR-amplified Adh genes.

The rice genus (Oryza L.) consists of 24 species with 10 recognized genome types. With the realization of many useful genes in species of wild rice, continuous efforts have been made to understand their genomic composition and relationships. However, the identification of rice genomes has often been difficult owing to complex morphological variation and formation of allotetraploids. Here we propose a rapid and reliable method for identifying rice genomes based on the restriction sites of PCR-amplified Adh genes. The experimental procedure was as follows: (i) amplify a portion of Adh1 and Adh2 genes with the locus-specific PCR primers; (ii) digest PCR products with restriction enzymes that distinguish different genomes; and (iii) run the digested products on 1.4% agarose gel, and photograph. Using various combinations of restriction digestion of the two Adh genes, all of the rice genomes can be identified.     

黑麂MHC - DQA2 基因多态性

利用牛特异性扩增DQA2 第2 外显子的嵌套引物,对黑麂基因组DNA 进行PCR 扩增和克隆测序,基于该
序列设计出黑麂DQA2 基因第2 外显子特异性引物。利用该引物,通过PCR - SSCP 以及克隆测序技术,从40 个
黑麂样品中获得4 个不同的DQA2 等位基因。没有一个个体同时具有2 个以上的等位基因,所有序列均不含插入
或缺失突变,不含终止密码子,因此,本研究所扩增的DQA2 基因可能是表达的单基因座位。抗原结合区(Peptide
binding region,PBR)非同义替换率(dn)显著大于同义替换率(ds) (P <0.05),暗示该座位曾经历过明
显的正选择作用;进一步利用CODEML 程序中的相关模型以及贝叶斯法检测出4 个受选择作用的氨基酸位点
(α11、α58、α62、α66),这4 个位点均位于PBR 区。基于NJ 法构建的部分偶蹄类DQA 外显子2 系统发生关系
显示,黑麂4 个DQA2 等位基因与牛、羊以及梅花鹿的DQA2 等位基因构成独立的进化枝,在该进化枝内,黑麂
DQA2 等位基因优先与牛DQA2 等位基因聚类,暗示黑麂DQA2 基因在进化过程中存在跨物种进化现象。上述结
果表明平衡选择是维持黑麂DQA2 基因多态性的主要机制。然而,本研究从40 个样品中仅检测出2 个杂合子,
黑麂DQA2 等位基因之间的频率存在显著差异,推测可能是所检测的样品来源于不同种群,由于华伦德效应
(The Whalund effect)导致杂合度降低,也不排除本文所设计的引物在PCR 扩增过程中存在无效等位基因。     

Optimization of RAPD‐PCR conditions in cattle

Abstract

Random Amplified Polymorphic DNA polymerase chain reaction (RAPD‐PCR) is a fast and easy way of identifying DNA polymorphisms generated from several regions of the genome. This could expedite the process of identifying informative polymorphic markers that may be linked to important genes controlling economic traits. In cattle, failure to obtain consistent amplification patterns in RAPD‐PCR has been a cause for concern. This has been attributed to the fact that decamer primers that are used in RAPD‐PCR reactions are likely to amplify regions of DNA where the primer‐template base pairing has some degree of mismatch and that these mismatches fail to repeat from reaction to reaction. This paper describes the use of tricine buffer along with changes in reaction components and thermal cycling conditions that has yielded consistent and reproducible RAPD‐PCR amplifications using single primers and double primer combinations on bovine DNA.     

Conserved and non-conserved loci of the glucagon gene in old world ruminating ungulates

The homology and diversification of genomic sequence encoding glucagon gene among native Egyptian buffalos, camel and sheep were tested using cattle as model. Oligodeoxynucleotide primers designed from the available GenBank data were used for PCR probing of the glucagon gene encoding sequence at different loci. The DNA oligomer probes were constructed to flank either the whole gene encoding sequence or different intra-gene encoding sequences. The PCR products were visualized using agarose gel electrophoresis. All species showed a same size band of prepro-glucagon when PCR was used to amplify the whole gene encoding sequence. In contrary, amplifications of different intra-gene loci failed to give the same results. The results indicated variable degrees of diversity among old world ruminating ungulates in the glucagon gene encoding sequence. Compared with other ruminants, the variation appears predominantly in camel. Surprisingly, the similarity in size between both amplification products of whole gene encoding sequence and the proposed size of glucagon cDNA definitely excludes the possibility of large intervening introns spanning the genomic sequence of the glucagon gene in these species. This indicates that, in contrast to other tested mammals, the glucagon gene includes an essentially full-length copy of glucagon mRNA. The study revealed a possible new aspect of glucagon gene evolution in order to correlate its corresponding protein function among different ruminant species.     

Hypoxia-Inducible Factor in ringed seal (Phoca hispida) tissues

Tissue hypoxia and ischemia-reperfusion pose a dangerous situation for oxidative stress. However, diving mammals and birds show pronounced resistance to oxidative injury under such conditions, which are a consequence of selective vasoconstriction during a dive. As the function of Hypoxia-Inducible Factor-1alpha (HIF-1alpha) in protection against and adaptation to hypoxia has been recognized in terrestrial animals, we have investigated the genomics and expression of this protein in ringed seal (Phoca hispida) in order to determine if it may play a protective role in this diving mammal. PCR studies using primers based on sequences from mouse HIF-1alpha exons 3, 4, 5, 6, 9, 10, 11, 12 and 15 showed that DNA from seal lung generated PCR products similar to those from mouse DNA. These studies have established that a putative HIF-1alpha gene exists in the seal genome that appears to have a similar but not identical sequence to the mouse gene. Seal lung and skeletal muscle tissues showed the highest relative levels of HIF-1alpha protein expression, with heart muscle showing significantly lower levels, and levels of HIF-1beta protein expression paralleled this situation. Analysis of oxidized cellular protein levels indicated that seal lung and heart muscle had the lowest levels of oxidized proteins. Thus, as seal lung tissue had the highest level of HIF-1alpha protein expression and the second lowest level of protein oxidation, this suggests that HIF-1alpha expression may have an important protective effect in this tissue in diving mammals. Our results support the hypothesis that HIF-1alpha expression is dependent on both tissue-specific energy requirements and adequate metabolic supply-to-demand ratio. Combined, the evidence available suggests that diving mammals have an overall anticipatory response to avoid the ill effects of dive-associated ischemia-reperfusion which may involve the HIF-1 system.