Characterization of Bovidae sex-determining gene SRY

In mammals, testis determination is under the control of the sex-determining gene SRY. This Y-linked gene encodes a protein with a DNA binding domain similar to those found in high-mobility-group proteins. Here we report the cloning and sequences of the SRY genes of yak and Chinese native cattle. Our data show that SRY genes in Bovidae are less divergent, especially in the coding and 3'' regions.     

Nuclear localization of the testis determining gene product SRY

We have studied the expression of the human SRY protein (termed p27SRY) in two different cell lines by using specific antibodies. Confocal microscopy enabled us to localize p27SRY precisely in the nucleus in a discrete punctuate pattern. Furthermore, through microinjection experiments, we have demonstrated that the localization of the p27SRY protein into the nucleus was an event involving the NH2-terminal part of the high mobility group (HMG) domain. With the help of several synthetic peptides and various p27SRY mutants, we have characterized a bipartite basic motif in this part of the protein corresponding to a nuclear localization signal. This nuclear localization signal appears to be highly conserved in SRY box- and HMB box-containing proteins, suggesting common properties of nuclear targeting within the HMG box protein family.     

Studies on the phylogenetic distribution of pyruvate oxidoreductases

Lactate dehydrogenase is present in all phyla examined. Alanopine and strombine dehydrogenase activities are present in all major phyla except the Arthropoda, Echinodermata and Chordata. Octopine dehydrogenase is also absent from the latter phyla and virtually absent from the Annelida. The opine dehydrogenase activities are generally absent from non-marine species. Relationships can be observed between malate dehydrogenase and glutamic-oxaloacetic transaminase and between the latter and glutamic-pyruvic transaminase in certain phyla. The results support the suggestion that the opine pathways are used in tissues to generate high (relatively) rates of energy production.     

水牛SRY基因的克隆及序列分析

根据荷斯坦牛SRY基因设计一对引物,采用聚合酶链式反应（PCR）技术,以中国沼泽型水牛（Swamp Buffalo）基因组DNA为模板,扩增得到SRY（Sex Deterimation region of Y chromosome）全序列约2005bp,其中1-504bp为5’启动子区,1196-2005bp为3’侧翼序列,在505-1195bp为SRY的外显子,编码229个氨基酸。在SRY HMG box区域设计探针,用地高辛标记后分别与雄性、雌性水牛基因组DNA进行Southern 杂交,结果显示该段序列只在雄性DNA样本中有杂交信号,证明SRY基因为雄性特异。BLAST比对结果显示与牛属动物SRY基因的同源性为96％,其中SRY基因HMG box区域同源性高达99%,说明SRY基因具有高度的进化保守性     

Evolution of the male-determining gene SRY within the cat family Felidae

In most placental mammals, SRY is a single-copy gene located on the Y chromosome and is the trigger for male sex determination during embryonic development. Here, we present comparative genomic analyses of SRY (705 bp) along with the adjacent noncoding 5' flank (997 bp) and 3' flank (948 bp) in 36 species of the cat family Felidae. Phylogenetic analyses indicate that the noncoding genomic flanks and SRY closely track species divergence. However, several inconsistencies are observed in SRY. Overall, the gene exhibits purifying selection to maintain function (omega = 0.815) yet SRY is under positive selection in two of the eight felid lineages. SRY has low numbers of nucleotide substitutions, yet most encode amino acid changes between species, and four different species have significantly altered SRY due to insertion/deletions. Moreover, fixation of nonsynonymous substitutions between sister taxa is not consistent and may occur rapidly, as in the case of domestic cat, or not at all over long periods of time, as observed within the Panthera lineage. The former resembles positive selection during speciation, and the latter purifying selection to maintain function. Thus, SRY evolution in cats likely reflects the different phylogeographic histories, selection pressures, and patterns of speciation in modern felids.     

A heuristic approach of maximum likelihood method for inferring phylogenetic tree and an application to the mammalian SOX-3 origin of the testis-determining gene SRY

Applying the tree bisection and reconnection (TBR) algorithm, we have developed a heuristic method (maximum likelihood (ML)-TBR) for inferring the ML tree based on tree topology search. For initial trees from which iterative processes start in ML-TBR, two cases were considered: one is 100 neighbor-joining (NJ) trees based on the bootstrap resampling and the other is 100 randomly generated trees. The same ML tree was obtained in both cases. All different iterative processes started from 100 independent initial trees ultimately converged on one optimum tree with the largest log-likelihood value, suggesting that a limited number of initial trees will be quite enough in ML-TBR. This also suggests that the optimum tree corresponds to the global optimum in tree topology space and thus probably coincides with the ML tree inferred by intact ML analysis. This method has been applied to the inference of phylogenetic tree of the SOX family members. The mammalian testis-determining gene SRY is believed to have evolved from SOX-3, a member of the SOX family, based on several lines of evidence, including their sequence similarity, the location of SOX-3 on the X chromosome and some aspects of their expression. This model should be supported directly from the phylogenetic tree of the SOX family, but no evidence has been provided to date. A recently published NJ tree shows implausibly remote origin of SRY, suggesting that a more sophisticated method is required for understanding this problem. The ML tree inferred by the present method showed that the SRYs of marsupial and placental mammals form a monophyletic cluster which had diverged from the mammalian SOX-3 in the early evolution of mammals.     

A network perspective on the topological importance of enzymes and their phylogenetic conservation

Background  

A metabolic network is the sum of all chemical transformations or reactions in the cell, with the metabolites being interconnected by enzyme-catalyzed reactions. Many enzymes exist in numerous species while others occur only in a few. We ask if there are relationships between the phylogenetic profile of an enzyme, or the number of different bacterial species that contain it, and its topological importance in the metabolic network. Our null hypothesis is that phylogenetic profile is independent of topological importance. To test our null hypothesis we constructed an enzyme network from the KEGG (Kyoto Encyclopedia of Genes and Genomes) database. We calculated three network indices of topological importance: the degree or the number of connections of a network node; closeness centrality, which measures how close a node is to others; and betweenness centrality measuring how frequently a node appears on all shortest paths between two other nodes.     

Organization of butyrate synthetic genes in human colonic bacteria: phylogenetic conservation and horizontal gene transfer

Butyrate producers constitute an important bacterial group in the human large intestine. Butyryl-CoA is formed from two molecules of acetyl-CoA in a process resembling beta-oxidation in reverse. Three different arrangements of the six genes coding for this pathway have been found in low mol% G+C-content gram-positive human colonic bacteria using DNA sequencing and degenerate PCR. Gene arrangements were strongly conserved within phylogenetic groups defined by 16S rRNA gene sequence relationships. In the case of one of the genes, encoding beta-hydroxybutyryl-CoA dehydrogenase, however, sequence relationships were strongly suggestive of horizontal gene transfer between lineages. The newly identified gene for butyryl-CoA CoA-transferase, which performs the final step in butyrate formation in most known human colonic bacteria, was not closely linked to these central pathway genes.     

Mapping the SRY gene in Microtus cabrerae: a vole species with multiple SRY copies in males and females.

The SRY gene is a single-copy, male-specific gene, located on the Y chromosome in most mammals. However, recently we have described the presence of multiple polymorphic copies of this gene in both males and females of the vole species Microtus cabrerae. Here, we present the chromosomal localization of SRY gene copies in this species by fluorescent in situ hybridization (FISH). This technique localized these gene copies in the short arm, and hence in the euchromatic region, of the Y chromosome. Furthermore, several copies of the SRY gene are located on the X chromosome. These copies are spread along the entire heterochromatic region of the X chromosome, occupying the whole short arm, the centromeric region, and the pericentromeric region of the long arm.     

The ontogeny and phylogenetic conservation of MAP 2 forms

Summary In the adult rat brain, MAP 2 is a high-molecular weight protein that is highly concentrated in dendrites. Immunoblots of homogenates of developing rat brain have indicated that a low-molecular weight form of MAP 2, MAP 2 c, is transiently expressed as the brain is undergoing morphogenesis. Using MAP 2-specific monoclonal antibodies, we have demonstrated that the compartmentalization of high-molecular weight MAP 2 and the developmental regulation of MAP 2 are conserved in mammalian, avian, and amphibian brain. We have also determined the distribution of MAP 2 c in developing neuronal tissue. MAP 2 c appears before high-molecular weight MAP 2 in developing neurons, and in contrast to the dendrite-specific high-molecular weight forms of MAP 2, MAP 2 c is present in axons and glia. We have also shown that MAP 2 c is present in the adult rat retina, where it is concentrated in regenerative photosensitive cells. The transient expression of MAP 2 c in the developing brain of three species as well as in adult photosensitive cells suggests a role for this protein in neurite growth and plasticity.Abbreviations MAP microtubule-associated protein - E embryonic day - P postnatal day     

Intergeneric complementation of a circadian rhythmicity defect: phylogenetic conservation of structure and function of the clock gene frequency.

The Neurospora crassa frequency locus encodes a 989 amino acid protein that is a central component, a state variable, of the circadian biological clock. We have determined the sequence of all or part of this protein and surrounding regulatory regions from additional fungi representing three genera and report that there is distinct, preferential conservation of the frequency open reading frame (ORF) as compared with non-coding sequences. Within the coding region, many of the domain hallmarks of the N. crassa protein are highly conserved, especially an internal region bearing the causative mutations in frq1 and frq7, the most extreme alleles in the frequency allelic series. Despite considerable diversity among the strains analyzed in terms of morphology, growth, circadian clock output and frq sequence, the ORF from the most distantly related fungus included in this study (Sordaria fimicola) rescues rhythmicity in a N.crassa frequency null strain. Both sequence conservation, and the ability of frequency from a genus displaying one developmental program to complement circadian defects in a separate genus with a distinct, clock-regulated developmental program, are consistent with a central role of the frequency gene product in a general circadian oscillator capable of controlling diverse outputs in a variety of systems.     

Identification of the horizontal gene transfer based on phylogenetic data

We suggest a new procedure to search for the genes with horizontal transfer events in their evolutionary history. The search is based on analysis of topology difference between the phylogenetic trees of gene (protein) groups and the corresponding phylogenetic species trees. Numeric values are introduced to measure the discrepancy between the trees. This approach was applied to analyze 40 prokaryotic genomes classified into 132 classes of orthologs. This resulted in a list of the candidate genes for which the hypothesis of horizontal transfer in evolution looks true.     

A familial mutation in the testis-determining gene SRY shared by both sexes

A familial mutation in SRY, the gene coding for the testis-determining factor TDF, was identified in an XY female with gonadal dysgenesis, her father, her two brothers and her uncle. The mutation consists of a T to C transition in the region of the SRY gene coding for a protein motif known as the high mobility group (HMG) box, a protein domain known to confer DNA-binding specificity on the SRY protein. This point mutation results in the substitution, at amino acid position 109, of a serine residue for phenylalanine, a conserved aromatic residue in almost all HMG box motifs known. This F109S mutation was not found in 176 male controls. When recombinant wildtype SRY and SRY^F109S mutant protein were tested in vitro for binding to the target site AAC AAAG, no differences in DNA-binding activity were observed. These results imply that the F109S mutation either is a rare neutral sequence variant, or produces an SRY protein with slightly altered in vivo activity, the resulting sex phenotype depending on the genetic back-ground or environmental factors.This paper is dedicated by G. S. to Professor Ulrich Wolf on the occasion of his 60th birthday     

Cloning the swamp buffalo SRY gene for embryo sexing with multiplex-nested PCR

The polymerase chain reaction (PCR) is an efficient method for sexing embryos. The objective of this study was to develop an accurate and reliable method for sexing swamp buffalo (Bubalus bubalis) embryos. The SRY gene from swamp buffalo genomic DNA was amplified by PCR, using primers based on the sequence of the Holstein SRY gene. This fragment was sequenced based on a BLAST search; the SRY gene was highly conserved. Using a Southern blot, there was a strong signal in genomic DNA only from male swamp buffalo. Two pairs of nested primers, targeted to amplify the swamp buffalo SRY conserved region, were designed for sex identification. Simultaneously, the G3PDH gene was co-amplified to serve as an internal control. A multiplex-nested PCR system was optimized by varying the following individually: concentrations of Mg(2+) and dNTPs, ratio of concentrations of primers and numbers of cycles. Biopsies of 27 IVF-derived embryos and 24 embryos fertilized with Y-chromosome-bearing sperm were examined. Using optimized procedures, clear signals following PCR amplification were obtained from all embryo samples; PCR amplification accuracy was further verified by comparing PCR and dot blots. We concluded that this PCR technique was highly reliable for sexing swamp buffalo embryos.     

A phylogenetic analysis based on the gene encoding phosphoglycerate kinase

Summary We have determined the nucleotide sequence of both genomic and complementary DNA (cDNA) for the gene encoding the glycolytic enzyme phosphoglycerate kinase from the ciliated protozoan Tetrahymena thermophila. The amino acid sequence for the enzyme has also been derived from the cDNA sequence. The gene contains an open reading frame of 1260 nucleotides encoding 420 amino acids. Coding sequence in genomic DNA is interrupted by two introns at positions corresponding to introns 3 and 4 in mammalian phosphoglycerate kinase genes. The derived amino acid sequence was used to prepare a phylogeny by aligning the Tetrahymena sequence with 25 other phosphoglycerate kinase amino acid sequences. The Tetrahymena sequence is a typical eukaryotic sequence. There is recognizable and clear homology across species that cover nearly the complete range of life forms. The phylogenetic reconstruction of these sequences generally supports the conclusions that have been reached using rRNA sequences.Offprint requests to: R.E. Pearlman