G23 Sequences Research Articles

Gallus gallus aggrecan gene-based phylogenetic analysis of selected avian taxonomic groups

Mitochondrial DNA (mtDNA) sequences remain the most widely used for phylogenetic analysis in birds. A major limitation of mtDNA sequences, however, is that mitochondria genes are inherited as a single linkage group. Here we describe the use of a 540-bp DNA sequence corresponding to the G3 domain of Gallus gallus nuclear aggrecan gene (AGCI) for phylogenetic analysis of the main groups of Galliformes including Phasianidae, Numididae, and Odontophoridae. We also included species from Cracidae and Megapodiidae which are considered by some as Craciformes and others, including here as Galliformes. The uncorrected sequence divergence of the G3 fragments ranges from 1% among the grouses to 36% between some of the distant groups within Galliformes. These sequences contain 39-48% AT nucleotides and the ratios of transition versus transversion are above 1.5 in majority of the comparisons. Using G3 sequences from an Anseriform, Oxyura jamaicensis, as out-groups, phylogenetic trees were obtained using maximum parsimony and distance algorithms and bootstrap analyses. These trees were consistent with those described using Avian sarcoma and leucosis virus gag genes and those from amino acid sequences of hemoglobin and lysozyme c. Our data also support relationships among Galliformes which were defined using mtDNA sequences. In addition to the general support of the five main families of Galliformes, our data are also consistent with previous work that showed Francolinus africanus and Gallus gallus are in the same clade and that Tetraoninae is a well-supported monophyletic subfamily within Phasianidae. The results presented here suggest that the AGC1 sequences meet the criterion of novel nuclear DNA sequences that can be used to help resolve the relationships among Galliformes.

Binding of HNF-3α in nuclear extracts of pancreatic islet α-cell line to the novel HNF-3 site-A in the glucagon gene

The pancreatic islet hormone glucagon stimulates hepatic glucose production and thus maintains blood glucose levels in the fasting state. Transcription factors of the hepatocyte nuclear factor-3 (HNF-3, also called Foxa) family are expressed in endoderm-derived tissues such as liver and pancreatic islets and, as part of their metabolic control, are required for cell-specific activation of the glucagon gene in pancreatic islet α-cells. However, their action at the glucagon gene is poorly understood. We have shown previously that the well characterized HNF-3 binding site in the G2 enhancer element of the rat glucagon gene is not conserved in humans and that the human G2 sequence lacks basal enhancer activity. However, a novel HNF-3 site (called site-A) has been identified that is conserved in rat, mouse, and humans and that mediates an activation of the glucagon gene by HNF-3 proteins expressed in an heterologous cell line. In the present study, a molecular characterization using protein-DNA binding and transient transfection assays revealed that the novel HNF-3 site-A confers cell-specific promoter activity in glucagon-producing pancreatic islet α-cell lines (InR1G9, αTC2). In contrast to other HNF-3 binding sites in the glucagon promoter that bind nuclear HNF-3β, the novel HNF-3 site-A was found to bind preferentially HNF-3α in nuclear extracts of a glucagon-producing pancreatic islet α-cell line. Mice with a targeted disruption of the HNF-3α gene have been shown to develop normally but exhibit neonatal growth retardation and die postnatally prior to 4 weeks of age, exhibiting hypoglycemia associated with inappropriately low levels of circulating glucagon. The results of the present study offer a mechanism that explains the decrease in glucagon gene expression in HNF-3α-deficient mice. The novel HNF-3 site-A is located just upstream of the TATA box (between -30 and -50) suggesting a role for HNF-3 proteins in addition to direct transcriptional activation

Genetic diversity and population dynamics of cyanophage communities in the Chesapeake Bay

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 34:105-116 (2004) - doi:10.3354/ame034105 Genetic diversity and population dynamics of cyanophage communities in the Chesapeake Bay Kui Wang, Feng Chen* Center of Marine Biotechnology, University of Maryland Biotechnology Institute, 701 East Pratt Street, Suite 266, Baltimore, Maryland 21202, USA *Corresponding author. Email: chenf@umbi.umd.edu ABSTRACT: In order to understand the genetic diversity and population dynamics of cyanophages in estuarine waters, the viral capsid assembly (g20) gene was used as a gene marker to monitor genetic variations of natural cyanomyovirus communities in the Chesapeake Bay, USA. Unique and diverse g20 sequences were found. Only 1 of 15 g20 genotypes was closely related to the known cyanomyovirus isolates. Most of the g20 genotypes in the bay were not related to the g20 clonal sequences recovered from open-ocean waters. Terminal-restriction fragment length polymorphism (T-RFLP) based on the g20 gene was developed to investigate spatial and temporal distribution of cyanomyovirus communities in the bay. The T-RFLP profiles of the g20 gene demonstrated that the cyanomyovirus population structures in the bay were more dynamic seasonally than spatially. Seasonal variation in the cyanophage community appeared to correspond to changes in host-cell density, which in turn was mainly affected by water temperature. This study represents the first effort to monitor both cyanophage titer and genetic diversity over time and space. The results of our study suggest that cyanophages could play a significant role in regulating Synechococcus biomass and population structure in the Chesapeake Bay. KEY WORDS: Cyanophage · Synechococcus · Natural virus community · Phylogenetic diversity · T-RFLP Full article in pdf format NextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 34, No. 2. Online publication date: February 04, 2004 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2004 Inter-Research.

Genetic diversity and temporal variation in the cyanophage community infecting marine Synechococcus species in Rhode Island's coastal waters.

The cyanophage community in Rhode Island's coastal waters is genetically diverse and dynamic. Cyanophage abundance ranged from over 10(4) phage ml(-1) in the summer months to less then 10(2) phage ml(-1) during the winter months. Thirty-six distinct cyanomyovirus g20 genotypes were identified over a 3-year sampling period; however, only one to nine g20 genotypes were detected at any one sampling date. Phylogenetic analyses of g20 sequences revealed that the Rhode Island cyanomyoviral isolates fall into three main clades and are closely related to other known viral isolates of Synechococcus spp. Extinction dilution enrichment followed by host range tests and PCR restriction fragment length polymorphism analysis was used to detect changes in the relative abundance of cyanophage types in June, July, and August 2002. Temporal changes in both the overall composition of the cyanophage community and the relative abundance of specific cyanophage g20 genotypes were observed. In some seawater samples, the g20 gene from over 50% of isolated cyanophages could not be amplified by using the PCR primer pairs specific for cyanomyoviruses, which suggested that cyanophages in other viral families (e.g., Podoviridae or Siphoviridae) may be important components of the Rhode Island cyanophage community.

Phylogenetic Diversity of Marine Cyanophage Isolates and Natural Virus Communities as Revealed by Sequences of Viral Capsid Assembly Protein Gene g20

In order to characterize the genetic diversity and phylogenetic affiliations of marine cyanophage isolates and natural cyanophage assemblages, oligonucleotide primers CPS1 and CPS8 were designed to specifically amplify ca. 592-bp fragments of the gene for viral capsid assembly protein g20. Phylogenetic analysis of isolated cyanophages revealed that the marine cyanophages were highly diverse yet more closely related to each other than to enteric coliphage T4. Genetically related marine cyanophage isolates were widely distributed without significant geographic segregation (i.e., no correlation between genetic variation and geographic distance). Cloning and sequencing analysis of six natural virus concentrates from estuarine and oligotrophic offshore environments revealed nine phylogenetic groups in a total of 114 different g20 homologs, with up to six clusters and 29 genotypes encountered in a single sample. The composition and structure of natural cyanophage communities in the estuary and open-ocean samples were different from each other, with unique phylogenetic clusters found for each environment. Changes in clonal diversity were also observed from the surface waters to the deep chlorophyll maximum layer in the open ocean. Only three clusters contained known cyanophage isolates, while the identities of the other six clusters remain unknown. Whether or not these unidentified groups are composed of bacteriophages that infect different Synechococcus groups or other closely related cyanobacteria remains to be determined. The high genetic diversity of marine cyanophage assemblages revealed by the g20 sequences suggests that marine viruses can potentially play important roles in regulating microbial genetic diversity.

Generation of human Fab monoclonal antibodies against preS1 of hepatitis B virus using repertoire cloning.

Human monoclonal antibodies (MAbs) have considerable potential in the prevention and treatment of many viral diseases. A combinatorial antibody library of heavy (Fd)- and light-chain genes derived from peripheral blood lymphocytes of a volunteer with high antibody titer to preS1 of HBV was constructed and expressed on the surface of filamentous phages. The library contained 7 x 10(9) independent clones. A phage antibody population from the third panning against preS1 was converted to one expressing soluble Fabs by removal of the g3 sequences from the pComb3 phagemid vector and subsequent transformation into E. coli TG1 cells. Screening of the library led to the identification of two clones, 3DW and 8GW, showing high reactivity toward preS1. The authenticity of the Fabs was confirmed by immunoblot analysis which yielded approximately 60 and approximately 30 kDa bands under nonreducing and reducing conditions, respectively. The soluble Fabs of 3DW and 8GW exhibited relative affinities of 6 x 10(5) and 8 x 10(6) M(-1), respectively. The sequencing results demonstrate that all Fd sequences belong to subgroup II and all light chain sequences belong to subgroup I. There are differences in CDR length and composition, especially in the FW3 and CDR3 regions of the heavy- and light-chain genes. These human Fab MAbs specific to preS1, generated from a combinatorial library, represent prototypes of passive immunotherapy candidates for viral hepatitis B.

Conserved basic residues in the C-type lectin and short complement repeat domains of the G3 region of proteoglycans.

Aggrecan is the major proteoglycan of the extracellular matrix in cartilage. It contains two N-terminal globular regions, G1 and G2, and one C-terminal globular region, G3. G3 is implicated in the intracellular processing of aggrecan and contains a C-type lectin carbohydrate recognition domain (CRD), frequent occurrences of a C-terminal short complement repeat (SCR) domain, and occasionally an N-terminal epidermal growth factor domain. The CRD and SCR domains in 13 G3 sequences were each subjected to structural analysis. Alignment of 131 sequences from all seven groups in the CRD superfamily defined a consensus length of 136 residues, in which 32% of residues were conserved. Although the G3 CRD sequences agreed with this consensus, they also contained five fully conserved basic residues that are atypical of the CRD superfamily. Homology modelling showed that four of these residues are located on a surface region on the CRD that is separate from the Ca2+-binding residues involved in carbohydrate interactions. One conserved basic residue is identical in position with that of a conserved basic residue that mediates hyaluronate binding in the structurally related proteoglycan tandem repeat (PTR) domain in G1 and in link protein. The alignment of 13 G3 SCR sequences with 101 sequences in the SCR superfamily showed good agreement with conserved residues in the SCR superfamily. There are also five conserved basic residues in the G3 SCR that are atypical of the SCR superfamily, and homology modelling showed that all five were located on one surface of the SCR. It is concluded that both the CRD and SCR domains in G3 possess basic residues that are atypical of their superfamilies and might be related to function, and that the G3 CRD domain shows an evolutionary relationship to the PTR domain in G1.

A pancreatic islet cell-specific enhancer-like element in the glucagon gene contains two domains binding distinct cellular proteins.

Interactions of nuclear proteins with cis-control elements are involved in the programmed developmental expression of the islet polypeptide hormone genes. Three transcriptional control elements within 300 base pairs of the 5'-flanking region of the rat glucagon gene interact with regulatory cellular proteins and direct transcription only in glucagon-producing islet cells. Two islet cell-specific enhancer-like elements (G2, G3) act together with the glucagon promoter (including the G1 element), which confers A cell specificity of glucagon gene expression. In the present study, the G3 element was analyzed in detail by protein binding and in vivo and in vitro transcription assays. Mutational analyses showed that the sequence of the G3 element comprises two distinct protein-binding domains: a more upstream domain A (5'-CGCCTGA-3'), and a more downstream domain B (5'GATTGAAGGGTGTA-3'). Binding of proteins to these two domains is mutually exclusive. Domain A, but not domain B, is responsible for both functional protein binding and the enhancement of transcription from the glucagon or thymidine kinase gene promoter chloramphenicol acetyltransferase reporter gene transfected in vivo into glucagon-producing islet cells (InR1-G9) and transcribed in vitro in a HeLa cell-free transcription system. In islet cell extracts, the Southwestern blot technique labeled a protein of 45 kDa binding to domain A within G3. We conclude that although the G3 sequence contains two protein-binding motifs, the organization of the G3 enhancer-like element is not bipartite. The islet cell specificity of the G3 element is conferred by a tissue-specific transcription factor or protein complex interacting with domain A of G3. This protein or protein complex recognizes different DNA sequences and provides promoter as well as enhancer activity because it binds also to the apparently unrelated sequence of the G1 promoter element.

Hantaan virus MRNA: Coding strategy, nucleotide sequence, and gene order

The M genome segment of Hantaan virus was molecularly cloned and the nucleotide sequence of cDNA was determined. The virion RNA is 3616 bases long with 3′- and 5′-terminal nucleotide sequences complementary for 18 bases. A single long open reading frame in the viral complementary-sense RNA had the potential to encode 1135 amino acids or a polypeptide of 126,000 Da. Amino-terminal sequences of isolated G1 and G2 envelope glycoproteins were determined, revealing a gene order with respect to message sense RNA of 5′-G1-G2–3′. Mature G1 begins 18 amino acids beyond the first AUG of the open reading frame, preceded by a short, hydrophobic leader sequence. G2 begins at the 649th amino acid of the open reading frame and also follows a hydrophobic sequence. Carboxy termini of G1 and G2 were localized and gene order was verified by immune precipitation of Hantaan proteins with antisera to synthetic peptides generated by using amino acid sequences derived from the cDNA sequence. The antipeptide sera were also reactive by immunoblotting with SDS-denatured G1 and G2. Molecular weights of 64,000 and 53,700 were calculated for the G1 and G2 glycoproteins, respectively, from their predicted amino acid sequences. Five potential asparagine-linked glycosylation sites were contained within the G1 amino acid sequence and two within the G2 sequence. These data are consistent with our previous estimates of the molecular weights and extent of glycosylation of the Hantaan envelope glycoproteins.