A solid phase plate assay for HIV-1 genotyping.

A prototype solid phase plate assay (SPPA) for the detection and genotyping of HIV-1 subtypes was developed using a PCR-based capture hybridization format. Well characterized HIV-1 reference controls of known subtypes and subtype specific capture oligonucleotide probes targeting several regions of the envelope (env) gene of HIV-1 were selected to develop the assay. The subtype specific oligonucleotide probes were covalently bound to microtubes in an ordered pattern and biotin labelled primers were used to amplify the target sequences. The PCR products were hybridized to the corresponding oligonucleotide probes, and colorimetrically detected by a chromogenic reaction using a standard microplate reader. All the HIV-1 subtype reference controls specifically hybridized to the corresponding capture probes and negligible cross-hybridization between subtypes was observed. To demonstrate the performance and reproducibility of the SPPA system and its validation with clinical samples, several human plasma samples of unknown and known HIV-1 subtypes were tested. The SPPA is highly specific and unambiguously identify the major subtypes of the HIV-1 M and O groups. This assay could be a useful method for subtyping samples of HIV-1 infected individuals and for disease management.

A duplex PCR assay for detection and genotyping of Herpes simplex virus in cerebrospinal fluid.

A duplex polymerase chain reaction (PCR) assay for the detection and genotyping of Herpes simplex virus (HSV) 1 and 2 from cerebrospinal fluid (CFS) of infants was developed. The glycoprotein D (gD) gene of HSV was selected as a target for amplification. The assay is highly specific, sensitive and reproducible. Herpes simplex virus detection is performed by agarose gel electrophoresis and Southern blot using a chemiluminescent probe. The probe hybridizes to sequences common to both HSV-1 and 2. A DNA fragment of HSV gD gene was cloned and used as positive control and to determine the specificity and sensitivity of the assay. The PCR assay is user-friendly and unambiguously differentiates in one-step both herpes virus strains. The assay is useful to screen CFS specimens from infants exposed to HSV during birth and at risk of developing encephalitis.

Production of a biologically active recombinant teleostean growth hormone in E. coli cells.

We have isolated and characterized several recombinant lambda phage clones carrying growth hormone (GH) cDNA of striped bass (Morone saxatilis). Nucleotide sequence and the predicted amino acid sequence of sbGH was determined from a recombinant clone carrying the longest cDNA insert. The sbGH cDNA encodes a pre-hormone of 204 amino acid residues. Comparison of the predicted amino acid sequence of sbGH with those of other vertebrates revealed different degrees of sequence identity: approximately 98% with European sea bass; 90% with bluefin tuna; bonito and red seabream; 71% with winter flounder; 64% with salmonids; 55% with carp; and 38% with human. Expression of the mature sbGH cDNA (without the signal peptide sequence) in E. coli cells under regulation of the lambda phage PL promoter produced a polypeptide of 20 kDa. Following renaturation, this recombinant hormone was shown to be biologically active in a radioreceptor competition binding assay and in the induction of hepatic insulin-like growth factor I (IGF-I) mRNA synthesis in vivo.

Gene transfer, expression and inheritance of pRSV-rainbow trout-GH cDNA in the common carp, Cyprinus carpio (Linnaeus).

A recombinant plasmid containing the Rous sarcoma virus-long terminal repeat (RSV-LTR) promoter linked to rainbow trout (Salmo gairdneri) growth hormone (GH) cDNA was microinjected into fertilized carp eggs. Genomic DNA extracted from pectoral fin of individual presumptive transgenic fish was analyzed by dot blot and Southern blot hybridization, using the RSV-LTR and/or the GH cDNA sequences as probes. Out of 365 presumptive transgenic fish analyzed, 20 individuals were found to contain pRSV-rtGH-cDNA sequence in the genomic DNA. Expression of the trout GH polypeptide was detected by immunobinding assay in the red blood cells of nine transgenic fish tested. The level of expression, however, varied among the transgenics and could not be correlated with exogenous DNA copy number. Although there was considerable variation in the sizes of the transgenic fish, those microinjected during the one-cell stage were (P less than 0.05) 22% larger, on the average, than their sibling controls. A randomly selected fraction of the progeny derived from crosses between transgenic males and non-transgenic females inherited the foreign DNA. These transgenic progeny grew faster (P less than 0.05) than their non-transgenic siblings.

Evolutionary implications of two rainbow trout growth hormone genes.

The primary structures of two rainbow trout growth hormone mRNAs (GH1 and GH2) have been deduced by direct sequencing of their respective cDNA clones and portions of the mRNA. Both GH1 and GH2 mRNA contain open reading frames comprised of 630 nucleotides and encode 210 amino acid residues of which 11 are variant. The translated regions of both mRNA are flanked by a short but rather conserved 5'-end, and a relatively long but highly diverged 3'-end. The differences at translated and 3'-untranslated regions suggest that the GH1 and GH2 mRNA originate from different loci. The GH1 and GH2 mRNA are likely transcribed from two distinct loci which were duplicated during tetraploidization of salmonid genome between 50 to 100 million years ago.The GH2 gene has been isolated and sequenced from a rainbow trout genomic library. This gene spans a region of approximately 4 kilobases. The trout GH gene is comprised of 6 exons and 5 introns, in contrast to 5 exons and 4 introns in mammals. The additional intron in the trout gene interrupts the translated regions that are analogous to the last exon of the mammalian counterpart. The alleged internally repeating sequences in mammalian GH, prolactin (Pr1) and placental lactogen (PL) are not observed in the predicted polypeptide sequence of trout GH. In addition, direct repeats that flank exons I, III and V of mammalian GH, Pr1 and PL genes are absent in trout gene. These findings indicate that the rainbow trout GH gene structure does not support the current hypothesis that internally repeated regions in GH, Pr1 and PL arose from a small primordial gene.

Molecular cloning and sequencing of coho salmon growth hormone cDNA.

A cDNA library was constructed using mRNA isolated from coho salmon pituitaries. By employing rainbow trout growth hormone cDNA as a probe, the coho salmon cDNA was isolated and the complete nucleotide (nt) sequence determined. The coding region contains 630 nt while the 5'- and 3'-untranslated regions are 64 and 489 nt in length, respectively. Comparison of the noncoding regions of coho and chum salmon cDNAs reveal identity at the 5' end but significant variation in the 3' end. Chum salmon and rainbow trout have identical amino acid (aa) sequences, but coho salmon growth hormone has a sequence that differs by 6 of the 188 predicted aa. Since salmonids are tetraploid, this difference may be the result of either divergence of the same growth hormone locus or of variation between different loci. Comparisons of the cDNA restriction maps of these three fish species suggest the former possibility.

A multilocus system for studying tissue and subcellular specialization. The pH and temperature dependence of the two major NADP-dependent isocitrate dehydrogenase isozymes of the fish Fundulus heteroclitus.

In the teleost fish Fundulus heteroclitus, there are three NADP-dependent isocitrate dehydrogenase isozymes. IDH-B2 is the only cytoplasmic isozyme, and IDH-C2 dominates the mitochondria of all tissues other than liver, where IDH-A2 is expressed. Since fish are ectotherms, their intracellular temperature and pH change directly with environmental temperature. In order to evaluate the influence of these environmental parameters on a model fish NADP-isocitrate dehydrogenase system, the major cytoplasmic (IDH-B2) and mitochondrial (IDH-C2) isozymes were kinetically evaluated as a function of pH and temperature. Whereas Vfmax and KmISOCm (where ISOC is isocitrate) were pH-independent, the Km for NADP was pH-dependent for both isozymes. The cytoplasmic isozyme (IDH-B2) had smaller KmNADP values between pH 7.0 and pH 8.0 than the mitochondrial form (IDH-C2). Vfmax and Km for substrate and coenzyme were temperature-dependent. Energy of activation for IDH-B2 and IDH-C2 was 10.6 and 12.8 kcal/mol, respectively. Both proteins had delta G not equal to values of about 15.8 kcal/mol, with significantly different distributions between delta H not equal to and delta S not equal to. The cytoplasmic isozyme (IDH-B2) appears to have a greater rate of catalysis than the mitochondrial enzyme (IDH-C2) at temperatures less than 30 degrees C. Moreover, the IDH-B2 isozyme had lower KmNADP values than the IDH-C2 isozyme at all temperatures, whereas the KmISOC values for the two isozymes were indistinguishable. Our data suggest that the two major NADP-dependent isocitrate dehydrogenase isozymes have unique physiological and metabolic functions that are adapted to the tissues and cellular compartments in which they are expressed.

A multilocus system for studying tissue and subcellular specialization. The three NADP-dependent isocitrate dehydrogenase isozymes of the fish Fundulus heteroclitus.

Three NADP-dependent isocitrate dehydrogenase isozymes in the teleost, Fundulus heteroclitus (L.), exhibit differences in tissue and subcellular distribution. These three proteins were purified and characterized as to native and subunit molecular weight, isoelectric pH, susceptibility to thermal denaturation, and certain kinetic parameters (Km and Vmax) for the oxidative decarboxylation of isocitrate at 25 degrees C and pH 7.4. The enzymes are dimers of 90 +/- 4 kDa with subunit molecular masses of 45 +/- 3 kDa. Isoelectric pH values were 7.00, 5.19, and 5.29 for IDH-A2, IDH-B2 and IDH-C2 (where IDH represents isocitrate dehydrogenase), respectively. While the monomer-dimer equilibrium is not influenced by substrates, the equilibrium appears to respond to buffer concentration and temperature. Enzyme activity is not affected upon dilution in the presence of buffer containing bovine serum albumin, however, its activity declines rapidly in the absence of bovine serum albumin. Thermal stability varies among the isozymes, and they do not denature by a simple first-order process. The presence of substrates, metal, and coenzymes independently provided enzyme stability, suggesting a random mechanism of substrate and cofactor binding. While IDH-A2 and IDH-B2 have identical KISOCm, IDH-B2 has a lower KNADPm. The most common mitochondrial isozyme (IDH-C2) has a greater KISOCm than either the less common mitochondrial isozyme (IDH-A2) or the cytoplasmic enzyme (IDH-B2). The KNADPm for IDH-C2 was the same as that of IDH-A2 but greater than that of IDH-B2. These Km differences are consistent with the cytoplasmic-mitochondrial shuttling of NADPH-reducing equivalents into the cytoplasm.