Biomedical subjects

A V Gordadze

Publications and source records attributed to A V Gordadze.

8 recordsLinked to original sources

PubMed2001

Notch1IC partially replaces EBNA2 function in B cells immortalized by Epstein-Barr virus.

Immortalization of B cells by Epstein-Barr virus (EBV) depends on the virally encoded EBNA2 protein. Although not related by sequence, the cellular Notch protein and EBNA2 share several biochemical and functional properties, such as interaction with CBF1 and the ability to activate transcription of a number of cellular and viral genes. Whether these similarities are coincidental or exemplify EBNA2 mimicry of evolutionarily conserved cellular signaling pathways is unclear. We therefore investigated whether activated forms of Notch could substitute for EBNA2 in maintaining the immortalized phenotype of EBV-infected B cells. To address this question, we devised a transcomplementation system using EREB2.5 cells. EREB2.5 cells are immortalized by EBV expressing a conditional estrogen receptor EBNA2 fusion protein (EREBNA2), and cellular proliferation is dependent on the availability of estrogen. Withdrawal of estrogen results in inactivation of EREBNA2, leading to growth arrest and eventually to cell death. Transduction of EREB2.5 cells with a lentiviral vector expressing wild-type EBNA2 rescued EREB2.5 cells from the growth-inhibitory effects of estrogen deprivation, in contrast to transduction with the lentivirus vector alone. EREB2.5 cells were also rescued by enforced expression of human Notch1IC after estrogen starvation, but this effect was restricted to cells expressing high levels of the transcription factor. Compared to wild-type EBNA2-expressing EREB2.5 cells, the Notch-expressing cells expanded more slowly after estrogen starvation, and once established, they continued to display a lower proliferation rate. Analysis of viral and cellular gene expression from transduced EREB2.5 cells after estrogen withdrawal indicated that both wild-type EBNA2- and Notch1IC-positive cells expressed c-Myc at levels similar to those found in parental EREB2.5 cells. However, the latter cells expressed LMP-1 far less efficiently than cells transduced with the wild-type EBNA2 gene. Cells rescued by either wild-type EBNA2 or Notch1IC expressed surface CD21 and CD23 proteins, but not CD10, indicating that induction of relevant type III latency markers was maintained. The data imply that both Notch and EBNA2 activate an important subset of cellular genes associated with type III latency and B-cell growth, while EBNA2 more efficiently induces important viral genes, such as LMP-1. Thus, exploitation of conserved Notch-related signaling pathways may represent a key mechanism by which EBNA2 contributes to EBV-induced cell immortalization.

B-Lymphocytes↗

PubMed2000

Sequence and functional analysis of EBNA-LP and EBNA2 proteins from nonhuman primate lymphocryptoviruses.

The Epstein-Barr virus (EBV) EBNA-LP and EBNA2 proteins are the first to be synthesized during establishment of latent infection in B lymphocytes. EBNA2 is a key transcriptional regulator of both viral and cellular gene expression and is essential for EBV-induced immortalization of B lymphocytes. EBNA-LP is also important for EBV-induced immortalization of B lymphocytes, but far less is known about the functional domains and cellular cofactors that mediate EBNA-LP function. While recent studies suggest that serine phosphorylation of EBNA-LP and coactivation of EBNA2-mediated transactivation are important, more detailed mutational and genetic studies are complicated by the repeat regions that comprise the majority of the EBNA-LP sequence. Therefore, we have used a comparative approach by studying the EBNA-LP homologues from baboon and rhesus macaque lymphocryptoviruses (LCVs) (baboon LCV and rhesus LCV). The predicted baboon and rhesus LCV EBNA-LP amino acid sequences are 61 and 64% identical to the EBV EBNA-LP W1 and W2 exons and 51% identical to the EBV EBNA-LP Y1 and Y2 exons. Five evolutionarily conserved regions can be defined, and four of eight potential serine residues are conserved among all three EBNA-LPs. The major internal repeat sequence also revealed a highly conserved Wp EBNA promoter with strong conservation of upstream activating sequences important for Wp transcriptional regulation. To test whether transcriptional coactivating properties were common to the rhesus LCV EBNA-LP, a rhesus LCV EBNA2 homologue was cloned and expressed. The rhesus LCV EBNA2 transcriptionally transactivates EBNA2-responsive promoters through a CBF1-dependent mechanism. The rhesus LCV EBNA-LP was able to further enhance rhesus LCV or EBV EBNA2 transactivation 5- to 12-fold. Thus, there is strong structural and functional conservation among the simian EBNA-LP homologues. Identification of evolutionarily conserved serine residues and regions in EBNA-LP homologues provides important clues for identifying the cellular cofactors and molecular mechanisms mediating these conserved viral functions.

Amino Acid Sequence↗

PubMed1999

Molecular cloning and expression of two hexamerin cDNAs from the mosquito, Aedes aegypti.

Fourth-instar larvae of Aedes aegypti synthesize two types of hexamerins, Hexamerin-1 (AaHex-1) and Hexamerin-2 (AaHex-2), whose subunits are distinguished by different methionine and aromatic amino acid contents. In early female pupae only the methionine-rich AaHex-1gamma subunit accumulates to two-fold higher levels than in males. To investigate the relationship between hexamerin structure and the roles of Hex-1 and Hex-2 during mosquito development and reproduction, we have cloned and sequenced cDNAs encoding the AaHex-2alpha, -2beta and AaHex-1gamma subunits. Comparison with other insect hexamerins revealed that the Aedes Hex-1 and Hex-2 proteins belong, respectively, to the two hexamerin subfamilies previously defined for brachyceran Diptera. Probes specific for the Hex-2alpha and Hex-1gamma transcripts showed that expression of both genes follows the same developmental timetable. However, greater Hex-1gamma mRNA accumulation may contribute to the higher levels of Hex-1 gamma protein in early female pupae.

Aedes↗

PubMed1999

Evolution of two types of rhesus lymphocryptovirus similar to type 1 and type 2 Epstein-Barr virus.

Rhesus monkeys and other nonhuman Old World primates are naturally infected with lymphocryptoviruses (LCV) that are closely related to Epstein-Barr virus (EBV). A rhesus LCV isolate (208-95) was derived from a B-cell lymphoma in a simian immunodeficiency virus-infected rhesus macaque. The EBNA-2 homologues from 208-95 and a previous rhesus LCV isolate (LCL8664) were polymorphic on immunoblotting, so the EBNA-2 genes from these two rhesus LCV were cloned, sequenced, and compared. The EBNA-2 genes have 40% nucleotide and 41% amino acid identities, and the differences are similar to those between the type 1 and type 2 EBV EBNA-2. Sequence from a portion of the LMP1 gene which is extremely divergent among different LCV was virtually identical between the 208-95 and LCL8664 strains, confirming a common rhesus LCV background. Thus, the EBNA-2 polymorphism defines the presence of two different rhesus LCV types, and both rhesus LCV types were found to be prevalent in the rhesus monkey population at the New England Regional Primate Research Center. The existence of two rhesus LCV types suggests that the selective pressure for the evolution of two LCV types is shared by human and nonhuman primate hosts.

Amino Acid Sequence↗

PubMed1997

Molecular cloning and expression of a hexamerin cDNA from the malaria mosquito, Anopheles gambiae.

During the last larval instar, dipteran insects synthesize two hexamerins rich in aromatic residues, typified by the larval serum proteins 1 and 2 (LSP-1 and LSP-2) of Drosophila melanogaster. We report here the characterization of a complete cDNA sequence encoding a LSP-1-like protein from a lower dipteran insect, the malaria mosquito Anopheles gambiae. The cDNA encodes the subunit of a homohexamer, A. gambiae hexamerin-1.1 (AgHex-1.1), which is a major pupal protein but only a minor constituent of late larval hemolymph. AgHex-1.1 is moderately rich in methionine (3.9%) and particularly rich in aromatic residues (21% Phe+Tyr). Cytogenetic analysis reveals AgHex-1.1 to be encoded by a single-copy gene localized to division 22F within the proximal 2La inversion breakpoint of chromosome 2 of A. gambiae. The AgHex-1.1 transcript is first detected in fourth-instar larvae (L4) and disappears abruptly in early pupae. In situ hybridization shows accumulation of the transcript uniquely in the larval fat body. AgHex-1.1 mRNA is re-expressed in male and female adults at about 10% of the L4 level, with no effect of bloodfeeding in females. The potential roles of AgHex-1.1 in Anopheles development and reproductive maturation are discussed.

Amino Acid Sequence↗

PubMed1997

Differential accumulation and tissue distribution of mosquito hexamerins during metamorphosis.

The pupal hexamerins were characterized for two mosquitoes representative of the culicine and anopheline families, Aedes aegypti and Anopheles gambiae. Like higher Diptera, both mosquito species express two types of hexamerins, Hex-1 and Hex-2, whose subunits are distinguished by different levels of methionine and aromatic amino acids. In A. aegypti there are two heterohexamers, AaHex-1 and AaHex-2. In A. gambiae there are two homohexamers, AgHex-1.1 and AgHex-1.2, and one heterohexamer, AgHex-2. These hexamerins are rich in aromatic residues, with 18-23% Phe + Tyr for Hex-1 subunits and 13-17% Phe + Tyr for Hex-2 subunits. In addition, both mosquito species synthesize methionine-rich Hex-1 subunits: Aedes AaHex-1 gamma (8% met) and Anopheles AgHex-1.1 (3.9% met). Aedes Hex-1 and Hex-2 proteins exhibit different, stage-specific tissue distributions: AaHex-2 is the primary hexamerin of late larval hemolymph whereas AaHex-1 is the most important non-hemolymph protein of early pupae. Although both proteins are stored in the pupal fat body, peak AaHex-1 levels are 2-fold higher. Both pupal protein levels decline rapidly between 25 and 36 h after pupation. Furthermore, AaHex-1 not only reaches peak values in female Aedes pupae later than in males, but the methionine-rich AaHex-1 gamma subunit level is specifically higher in females. These observations suggest different roles for Hex-1 and Hex-2 during mosquito development.

Aedes↗

PubMed1997

Mosquito hexamerins: characterization during larval development.

We report here the first examination of hexamerins expressed during mosquito larval development. Haemolymph proteins from fourth-instar larvae of six species representing the two major subfamilies of mosquitoes were characterized by immunoblotting using antisera to calliphorin, the major hexamerin of the blowfly. Calliphora vicina, or to LSP1 or LSP2, the two distinct hexamerins of Drosophila melanogaster. In each mosquito species the antisera demonstrated the presence of multiple abundant hexamerin polypeptides of 66-85 kDa in molecular weight. According to the subunit composition of native proteins, the larval hexamerins from both Aedes aegypti and Anopheles gambiae form heterohexamers. Furthermore, the two major Aedes hexamerin subunits (AaHex1 and AaHex2) are neither rich in aromatic amino acids nor methionine. cDNA clones encoding AaHex1 and AaHex2 were isolated and used to show that hexamerin mRNA is uniquely expressed in fourth-instar larvae of both A. aegypti and A. gambiae and disappears rapidly at the onset of pupal development.

Aedes↗

PubMed1996

Long PCR-based technique for detection of transposon insertions in and around cloned genes of Drosophila melanogaster.

A technique to detect a transposable element insertion greater than 5 kb away from a given gene-specific site is described. PCR is performed on genomic DNA isolated from a pool containing one heterozygous mutant fly, carrying an amplifiable allele, within a pool of 100 flies with no amplifiable sequences. A model procedure for optimizing PCR conditions and a test for primer ability to amplify sequences greater than 5 kb in length from very low dilutions of mutated sequences within non-amplifiable wild-type genomic DNA are described. The optimal annealing temperature range is shown to be as narrow as a 2 degrees C interval. Careful primer design is critical to the success of detection. Under the conditions developed, we detected many local transposable element hopping events, averaging about 3-4 per 50 flies, with the size of the PCR products being in the range of 100-6000 bp. In some cases, even larger (up to 8000 bp) bands were detected.

Animals↗