EST-based gene discovery in pig: virtual expression patterns and comparative mapping to human

A molecular understanding of porcine reproduction is of biological interest and economic importance. Our Midwest Consortium has produced cDNA libraries containing the majority of genes expressed in major female reproductive tissues, and we have deposited into public databases 21,499 expressed sequence tag (EST) gene sequences from the 3 end of clones from these libraries. These sequences represent 10,574 different genes, based on sequence comparison among these data, and comparison with existing porcine ESTs and genes indicate as many as 4652 of these EST clusters are novel. In silico analysis identified sequences that are expressed in specific pig tissues or organs and confirmed the broad expression in pig for many genes ubiquitously expressed in human tissues. Furthermore, we have developed computer software to identify sequence similarity of these pig genes with their human counterparts, and to extract the mapping information of these human homologues from genome databases. We demonstrate the utility of this software for comparative mapping by localizing 61 genes on the porcine physical map for Chromosomes (Chrs) 5, 10, and 14. The following Accession numbers were assigned to our deposited sequences: BF701840 – BF704551, BF708383, BF708386 – BF713604, BG322266 – BG322271, BI398567 – BI405235, BQ597354 – BQ605166.     

Tests of candidate genes in breed cross populations for QTL mapping in livestock

In recent years, several F₂ crosses between outbred lines of livestock have been developed to identify quantitative trait loci (QTL). These populations are valuable for further genetic analysis, including positional candidate gene loci (CGL). Analysis of CGL in F₂ populations is, however, hindered by extensive between-breed linkage disequilibrium (LD). The objectives here were to develop and evaluate three tests for CGL in simulated F₂ breed-cross populations. 1) A standard association test, based on the fixed effect of CGL genotype. This test was significant for CGL at considerable distances from the QTL. 2) A marker-assisted association test, based on a test at the CGL of the fixed effect of CGL genotype in a breed-cross QTL interval mapping model. This removed the impact of between-breed LD, but was not powerful in detecting CGL closely linked to the QTL, unless the CGL was the QTL. 3) An F-drop test, comparing F ratios for a QTL at the CGL with and without the CGL included as fixed effect. It had low power to distinguish close from distant CGL. Power to distinguish two CGL within 10 cM from the QTL was limited and little improved by including QTL effects associated with markers to remove between-breed LD, although the power was greater when one of the CGL was the causative mutation. Therefore, while we conclude that candidate gene tests in QTL mapping populations must be interpreted with caution, we now have a clearer picture of the value of candidate gene tests in these populations.     

High-Resolution Genetic Map of the Human Glutamyl Aminopeptidase Gene (ENPEP)

The murine B-lymphocyte differentiation antigen BP-1/6C3 has been identified as glutamyl aminopeptidase (EAP), the gene symbol for which isENPEP.Using genomic DNA encoding for human EAP as a probe, we identified theENPEPgene location on human chromosome 4q25 by polymerase chain reaction analysis of a human/rodent somatic cell hybrid mapping panel and by fluorescencein situhybridization. Using a radiation hybrid panel, the gene order aroundENPEPwas determined to be centromere–D4S1236–(570 kb)–ENPEP–(210 kb)–D4S262–(270 kb)–D4S953–(270 kb)–D4S474–(570 kb)–IF. The linkage ofENPEPto complement factor I (IF) confirms the human chromosome band 4q25 localization predicted from the chromosomal location of murineENPEP.HumanENPEPthus provides an additional marker for the long arm of chromosome 4 that should facilitate studies of this genomic region.     

Carbohydrate receptor-mediated gene transfer to human T leukaemic cells

The mucin-type carbohydrate Tn cryptantigen (GalNAc1-O-Ser/Thr,where GalNAc is N-acetyl-D-galactosamine) is expressed in manycarcinomas, in haemopoietic disorders including the Tn syndrome,and on human immunodeficiency virus (HIV) coat glycoproteins,but is not expressed on normal, differentiated cells becauseof the expression of a Tn-processing galactosyltransferase.Using Jurkat T leukaemic cells which express high levels ofTn antigen due to deficient Tn galactosylation, we have establishedthe Tn antigen-mediated gene transfer and demonstrate the considerableefficiency of this approach. We used poly(L-lysine) conjugatesof the monoclonal antibody 1E3 directed against the Tn antigento deliver the luciferase and ß-galactosidase reportergenes to Jurkat cells by receptor-mediated endocytosis. Additionof unconjugated 1E3 reduced transfection efficiency in a concentration-dependentmanner and incubation with free GalNAc abolished DNA transfercompletely, indicating that gene delivery is indeed mediatedby the Tn antigen. Pre-treatment of Jurkat cells with Vibriocholerae sialidase, which uncovers additional Tn antigens, resultedin an improvement of gene transfection. Both human and chickenadenovirus particles attached to the DNA/polylysine complexstrongly augmented transgene expression. When the ß-galactosidase(lacZ) gene was delivered to Jurkat cells by Tn-mediated endocytosis,up to 60% of the cells were positive in the cytochemical stainusing 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside(X-gal) as a chromogenic substrate. The efficiency of the transferrinreceptor-mediated DNA uptake into Jurkat cells was comparativelylow, although these cells were shown to express considerableamounts of transferrin receptor. We show here that a mucin-typecarbohydrate antigen mediates highly efficient DNA uptake byendocytosis into Jurkat T cells. This method represents a 50-foldimprovement of Jurkat cell transfection efficiency over otherphysical gene transfer techniques. Specific gene delivery toprimary cancer cells exhibiting Tn epitopes may especially bedesirable in immunotherapy protocols. adenovirus endocytosis gene transfer T cell Tn antigen     

In Vitro HIV-1 Evolution in Response to Triple Reverse Transcriptase Inhibitors & In Silico Phenotypic Analysis

Background

Effectiveness of ART regimens strongly depends upon complex interactions between the selective pressure of drugs and the evolution of mutations that allow or restrict drug resistance.

Methods

Four clinical isolates from NRTI-exposed, NNRTI-naive subjects were passaged in increasing concentrations of NVP in combination with 1 µM 3 TC and 2 µM ADV to assess selective pressures of multi-drug treatment. A novel parameter inference procedure, based on a stochastic viral growth model, was used to estimate phenotypic resistance and fitness from in vitro combination passage experiments.

Results

Newly developed mathematical methods estimated key phenotypic parameters of mutations arising through selective pressure exerted by 3 TC and NVP. Concentrations of 1 µM 3 TC maintained the M184V mutation, which was associated with intrinsic fitness deficits. Increasing NVP concentrations selected major NNRTI resistance mutations. The evolutionary pathway of NVP resistance was highly dependent on the viral genetic background, epistasis as well as stochasticity. Parameter estimation indicated that the previously unrecognized mutation L228Q was associated with NVP resistance in some isolates.

Conclusion

Serial passage of viruses in the presence of multiple drugs may resemble the selection of mutations observed among treated individuals and populations in vivo and indicate evolutionary preferences and restrictions. Phenotypic resistance estimated here “in silico” from in vitro passage experiments agreed well with previous knowledge, suggesting that the unique combination of “wet-” and “dry-lab” experimentation may improve our understanding of HIV-1 resistance evolution in the future.     

Associations of Alleles of the Esterase-1 Locus with Gene Arrangements of the Left Arm of the Second Chromosome in DROSOPHILA ROBUSTA

Evidence of strong associations of Est-1 alleles with the 2L, 2L1 and 2L3 gene arrangements of the left arm of the second chromosome in D. robusta is presented. Each gene arrangement is polymorphic for three to four Est-1 alleles. The allele frequencies differ in the 2L3 and 2L arrangements; the allele Est-1^.92 is 8% in the 2L3 arrangement (n=203)—this allele is 82% in the 2L arrangement (n=203); the allele Est-1^1.0 is 66% and 14.8% in the 2L3 and 2L arrangements, respectively. There are no differences in allele frequencies in 2L3 arrangements from any of the widely separated seven different populations; similarly the allele frequencies in the 2L arrangement are alike in all five widely separated populations studied. The allele frequencies in the 2L1 arrangement are intermediate to those observed in the 2L3 and the 2L arrangements and show north-south clinal change. These associations between Est-1 alleles and gene arrangements of the left arm of the second chromosome are due to natural selection favoring different allele frequencies in different gene arrangements, as a result of epistatic interactions between the Est-1 locus and the loci on the gene arrangements. As expected, we observe that the proportion of heterozygotes is greater in the inversion heterokaryotypes than in the homokaryotypes.