Comparative study and expression analysis of the interferon gamma gene locus cytokines in Xenopus tropicalis

Using bioinformatics approach, the genome locus containing interleukin (IL)-22, IL-26, and interferon gamma (IFN-gamma) genes has been identified in the amphibian, Xenopus tropicalis. Like that in other vertebrates such as fish, birds, and mammals, the Xenopus IL-22, IL-26, and IFN-gamma are clustered in the same chromosome and the adjacent genes are conserved. The genomic structures of the Xenopus IL-22, IL-26, and IFN-gamma gene were identical to that of their mammalian counterparts. The Xenopus IL-22 and IL-26 genes contained five exons and four introns while the Xenopus IFN-gamma gene consisted of four exons and three introns. The Xenopus IL-22, IL-26, and IFN-gamma share 14.1-41.6%, 14.6-31.2%, and 23.7-36.5% identity to their counterparts in other species, respectively. Reverse-transcription polymerase chain reaction (PCR) and real-time quantitative PCR analyses revealed that the expression of IL-22, IL-26, and IFN-gamma genes was significantly upregulated after simulation with bacterial polyliposaccharide and/or synthetic double-stranded poly(I:C), suggesting these cytokines like those in other vertebrates play an important role in regulating immune response in Xenopus.

Cloning, characterization and promoter analysis of common carp hairy/Enhancer-of-split-related gene, her6

Some members of hairy/Enhancer-of-split-related gene (HES) family have important effects on axial mesoderm segmentation and the establishment and maintenance of the somite fringe. In fishes. the her6 gene, a member of the HES family, is the homologue Of heS1 in mammals and chicken. In this study, the her6 gene and its full-length cDNA from the common carp (Cyprinus carpio) were isolated and characterized. The genomic sequence of common carp her6 is approximately 1.7 kb. with four exons and three introns, and the full-length cDNA of 1314 bp encodes a Putative polypeptide of 271 amino acids. To analyse the promoter sequence of common carp her6. sequences of various lengths upstream from the transcription initiation site of her6 were fused to enhanced green fluorescent. protein gene (eGFP) and introduced into zebrafish embryos by microinjection to generate transgenic embryos. Our results show that the upstream sequence of 500 bp can direct highly efficient and tissue-specific expression of eGFP in zebrafish embryos. whereas a fragment of 200 bp containing the TATA box and a partial suppressor of hairless paired site sequence (SPS) is not sufficient to drive eGFP expression in zebrafish embryos.

Gene structure and transcription of IRF-2 in the mandarin fish Siniperca chuatsi with the finding of alternative transcripts and microsatellite in the coding region

The gene of interferon regulatory factor-2 (IRF-2) has been cloned from the mandarin fish (Siniperca chuatsi). The IRF-2 gene has 6,418 nucleotides (nt) and contains eight exons and seven introns, encoding two mRNAs. The two IRF-2 mRNAs each contained an open reading frame of 873 nt, which both translate into the same 291 amino acids but differed in their 5' untranslated region: one mRNA was transcribed initially from the exon 1 bypassing exon 2, while the other was transcribed from the exon 2. The microsatellites (CA repeats) could be found in the carboxyl terminal region of mandarin fish IRF-2, which result in the truncated form molecules. The microsatellites' polymorphism was investigated, and eight alleles were found in 16 individuals. The microsatellites were also examined in IRF-2 of several freshwater perciform fishes. The transcription of the IRF-2 in different tissues with or without poly inosine-cytidine stimulation was analyzed by real-time PCR, and the constitutive transcription of both molecules could be detected in all the tissues examined.

Characterization of two genes encoding leucine-rich repeat-containing proteins in grass carp Ctenopharyngodon idellus

The cDNAs and genes of two different types of leucine- rich repeat-containing proteins from grass carp ( Ctenopharyngodon idellus) were cloned. Homology search revealed that the two genes, designated as GC-GARP and GC-LRG, have 37% and 32% deduced aminoacid sequence similarities with human glycoprotein A repetitions predominant precursor ( GARP) and leucine-rich alpha2-glycoprotein (LRG), respectively. The cDNAs of GC-GARP and GC-LRG encoded 664 and 339 amino acid residues, respectively. GC-GARP and GC-LRG contain many distinct structural and/or functional motifs of the leucine- rich repeat (LRR) subfamily, such as multiple conserved 11-residue segments with the consensus sequence LxxLxLxxN/CxL ( x can be any amino acid). The genes GC-GARP and GC-LRG consist of two exons, with 4,782 bp and 2,119 bp in total length, respectively. The first exon of each gene contains a small 5'-untranslated region and partial open reading frame. The putative promoter region of GC-GARP was found to contain transcription factor binding sites for GATA-1, IRF4, Oct-1, IRF-7, IRF-1, AP1, GATA-box and NFAT, and the promoter region of GC-LRG for MYC-MAX, MEIS1, ISRE, IK3, HOXA9 and C/EBP alpha. Phylogenetic analysis showed that GC-GARP and mammalian GARPs were clustered into one branch, while GC-LRG and mammalian LRGs were in another branch. The GC-GARP gene was only detected in head kidney, and GC-LRG in the liver, spleen and heart in the copepod ( Sinergasilus major)- infected grass carp, indicating the induction of gene expression by the parasite infection. The results obtained in the present study provide insight into the structure of fish LRR genes, and further study should be carried out to understand the importance of LRR proteins in host - pathogen interactions.

Development of polymorphic EST markers in Penaeus monodon: applications in penaeid genetics

The objective of this study was to develop type I markers for genome mapping and other genetic studies of Penaeus monodon. Primers were designed based on expressed sequence tags (ESTs) from a P monodon cephalothorax cDNA library to amplify 100-300 bp products. 34 of the primer pairs successfully amplified PCR products from genomic DNA. Single-strand conformation polymorphism analysis showed that similar to 30% of the ESTs tested exhibit polymorphism in a test panel of P monodon individuals. Mendelian inheritance of the EST-derived markers has been established in two international reference mapping families of P monodon, and mapping of these markers is in progress. Some ESTs were successfully amplified from other Penaeus species (P. chinensis, P japonicus and P vannamei), indicating that the markers are applicable in cross-species comparison. Two populations of P. japonicus could be differentiated using one of the ESTS. In conclusion, the polymorphic EST markers developed in this study are applicable in genome mapping and population genetic studies of penaeid shrimp. (C) 2002 Elsevier Science B.V. All rights reserved.

Comparative molecular population genetics of phycoerythrin locus in Prochlorococcus

As the only remainder type of phycobiliproteins in Prochlorococcus, the actual role of phycoerythrin still remains unknown. Previous studies revealed that two different forms of phycoerythrin gene were found in two ecotypes of Prochlorococcus that are specifically adapted to either high light (HL) or low light (LL) conditions. Here we analyze patterns of phycoerythrin nucleotide variation in the HL- and LL-Prochlorococcus populations. Our analyses reveal a significantly greater number of non-synonymous fixed substitutions in peB and peA than expected based on interspecific comparisons. This pattern of excess non-synonymous fixed substitutions is not seen in other five phycoerythrin-related genes (peZ/V/Y/T/S). Several neutrality statistical tests indicate an excess of rare frequency polymorphisms in the LL-Prochlorococcus data, but an excess of intermediate frequency polymorphisms in the HL-Prochlorococcus data. Distributions of the positively selected sites identified using the likelihood ratio test, when mapped onto the phycoerythrin tertiary structure, reveal that HL- and LL-phycoerythrin should be under different selective patterns. These findings may provide insights into the likely role of selection at the phycoerythrin locus and motivate further research to unveil the function of phycoerythrin in Prochlorococcus.

Population genetics of Crassostrea ariakensis in Asia inferred from microsatellite markers

Crassostrea ariakensis is an important aquacultured oyster species in Asia, its native region. During the past decade, consideration was given to introducing C. ariakensis into Chesapeake Bay, in the United States, to help revive the declining native oyster industry and bolster the local ecosystem. Little is known about the ecology and biology of this species in Asia due to confusion with nomenclature and difficulty in accurately identifying the species of wild populations in their natural environment. Even less research has been done on the population genetics of native populations of C. ariakensis in Asia. We examined the magnitude and pattern of genetic differentiation among 10 wild populations of C. ariakensis from its confirmed distribution range using eight polymorphic microsatellite markers. Results showed a small but significant global theta (ST) (0.018), indicating genetic heterogeneity among populations. Eight genetically distinct populations were further distinguished based on population pairwise theta (ST) comparisons, including one in Japan, four in China, and three populations along the coast of South Korea. A significant positive association was detected between genetic and geographic distances among populations, suggesting a genetic pattern of isolation by distance. This research represents a novel observation on wild genetic population structuring in a coastal bivalve species along the coast of the northwest Pacific.

Molecular genetics of the imprinted gene - SPROUTY3 (SPRY3)

Sprouty proteins are key regulators of cell growth and branching morphogenesis during development. Human SPRY3 which maps to the pseudoautosomal region 2, undergoes random X-inactivation in females and preferential Y-inactivation in males, behaving as though genetically X-linked. Spry3 is widely expressed in neuronal tissues, being found at high levels in the cerebellum and particularly in the Purkinje cells which, notably, are deficient in the autistic brain. Spry3 is also highly expressed in other ganglia in adults including retinal ganglion cells, dorsal root ganglion and superior cervical ganglion. SPRY3 enhancer can drive SPRY3 expression in the lung airway, which is consistent with a role in branching morphogenesis and the function of the original Drosophila Spry gene, which is critical for lung morphogenesis, providing a possible explanation for an observed anatomic abnormality in the autistic lung airway. In the human and mouse, the SPRY3 core promoter contains an AG-rich repeat and we found evidence of coexpression, promoter binding and regulation of SPRY3 expression by transcription factors EGR1, ZNF263 and PAX6. Spry3 over-expression in mouse superior cervical ganglion cells inhibits axon branching and Spry3 knockdown in those cells increases axon branching, consistent with known functions of other Sprouty proteins. Novel SPRY3 upstream transcripts that I characterised originate from three start sites in the X-linked F8A3 – TMLHE gene region, which is recently implicated in autism causation. Arising from these findings, I propose that the lung airway abnormality and low levels of blood carnitine found in autism suggest that deregulation of SPRY3 may underpin a subset of autism cases.

Use of pulsed field gel electrophoresis to characterize BCR gene involvement in CML patients lacking M-BCR rearrangement.

We studied the pattern of BCR involvement in 52 patients with chronic myeloid leukemia by Southern blotting. Of 33 Philadelphia (Ph)-positive patients, 30 had evidence of M-BCR rearrangement, two cases were difficult to interpret, and one clearly lacked evidence of M-BCR rearrangement. Of 19 Ph-negative patients, nine showed M-BCR rearrangement, nine showed no rearrangement, and one result was uncertain. We selected for more detailed study eight patients (three Ph-positive and five Ph-negative). Two of the Ph-positive patients, whose Southern blots were difficult to interpret, had rearranged bands when the BCR gene was studied by pulsed field gel electrophoresis (PFGE). Results of PFGE studies and in situ hybridization to metaphase chromosomes in the third Ph-positive patient, whose DNA clearly lacked M-BCR rearrangement on Southern analysis, were consistent with a breakpoint on chromosome 22 located 3' of all known exons of the BCR gene. However, mRNA studied with the polymerase chain reaction showed evidence of a classical b2-a2 linkage. The findings in this patient may be explained by an unusual genomic breakpoint downstream of the BCR gene associated with long range splicing that excluded all of the 3' BCR exons. Of the five patients with Ph-negative M-BCR non-rearranged CML studied by PFGE for BCR gene rearrangement, none had evidence of rearranged bands. We conclude that PFGE is a valuable adjunct to standard molecular techniques for the study of atypical cases of CML. Occasional patients with Ph-positive CML have breakpoints outside M-BCR. The BCR gene is probably not involved in patients with Ph-negative, M-BCR non-rearranged CML.

The characterization of twenty sequenced human genomes.

We present the analysis of twenty human genomes to evaluate the prospects for identifying rare functional variants that contribute to a phenotype of interest. We sequenced at high coverage ten "case" genomes from individuals with severe hemophilia A and ten "control" genomes. We summarize the number of genetic variants emerging from a study of this magnitude, and provide a proof of concept for the identification of rare and highly-penetrant functional variants by confirming that the cause of hemophilia A is easily recognizable in this data set. We also show that the number of novel single nucleotide variants (SNVs) discovered per genome seems to stabilize at about 144,000 new variants per genome, after the first 15 individuals have been sequenced. Finally, we find that, on average, each genome carries 165 homozygous protein-truncating or stop loss variants in genes representing a diverse set of pathways.

Host genetics and HIV-1: the final phase?

This is a crucial transition time for human genetics in general, and for HIV host genetics in particular. After years of equivocal results from candidate gene analyses, several genome-wide association studies have been published that looked at plasma viral load or disease progression. Results from other studies that used various large-scale approaches (siRNA screens, transcriptome or proteome analysis, comparative genomics) have also shed new light on retroviral pathogenesis. However, most of the inter-individual variability in response to HIV-1 infection remains to be explained: genome resequencing and systems biology approaches are now required to progress toward a better understanding of the complex interactions between HIV-1 and its human host.

Geography and Genetics of Ecological Speciation in Pea Aphids

During ecological speciation, divergent natural selection drives evolution of ecological specialization and genetic differentiation of populations on alternate environments. Populations diverging onto the same alternate environments may be geographically widespread, so that divergence may occur at an array of locations simultaneously. Spatial variation in the process of divergence may produce a pattern of differences in divergence among locations called the Geographic Mosaic of Divergence. Diverging populations may vary in their degree of genetic differentiation and ecological specialization among locations. My dissertation examines the pattern and evolutionary processes of divergence in pea aphids (Acyrthosiphon pisum) on alfalfa (Medicago sativa) and clover (Trifolium pretense). In Chapter One, I examined differences among North American aphid populations in genetic differentiation at nuclear, sequence-based markers and in ecological specialization, measured as aphid fecundity on each host plant. In the East, aphids showed high host-plant associated ecological specialization and high genetic differentiation. In the West, aphids from clover were genetically indistinguishable from aphids on alfalfa, and aphids from clover were less specialized. Thus, the pattern of divergence differed among locations, suggesting a Geographic Mosaic of Divergence. In Chapter Two, I examined genomic heterogeneity in divergence in aphids on alfalfa and clover across North America using amplified fragment length polymorphisms (AFLPs). The degree of genetic differentiation varied greatly among markers, suggesting that divergent natural selection drives aphid divergence in all geographic locations. Three of the same genetic markers were identified as evolving under divergent selection in the eastern and western regions, and additional divergent markers were identified in the East. In Chapter Three, I investigated population structure of aphids in North America, France, and Sweden using AFLPs. Aphids on the same host plant were genetically similar across many parts of their range, so the evolution of host plant specialization does not appear to have occurred independently in every location. While aphids on alfalfa and clover were genetically differentiated in most locations, aphids from alfalfa and clover were genetically similar in both western North America and Sweden. High gene flow from alfalfa onto clover may constrain divergence in these locations.