Structural and promoter regions of the murine pyruvate carboxylase gene.

We have cloned and sequenced the gene encoding mouse pyruvate carboxylase (mPC) [EC 6.4.1.1]. The coding region contains 19 exons, one 5'-untranslated region exon, and 19 introns in 22 kb of genomic DNA. This gene's exon/intron organization is highly conserved with respect to rat and human PC genes. The mPC gene promoter lacks canonical TATA and CCAAT boxes, in common with a number of housekeeping genes. Transient expressions in COS-1 of a luciferase reporter gene under the control of 5'-nested deletions of the 5'-flanking sequence of the mPC gene have identified the 166-bp minimal sequence required for basal transcription. Alternative splicing at the 5'-untranslated region exon of the mouse PC gene results in the production of two alternate transcripts bearing different 5'-noncoding regions. Both transcripts are highly expressed in kidney and liver and moderately expressed in heart and testis and expressed at a low level in spleen.

A non-stop, single-tube, semi-nested PCR technique for grading the severity of white spot syndrome virus infections in Penaeus monodon.

A single-tube, non-stop, semi-nested polymerase chain reaction (PCR) technique was developed for simultaneous detection and severity grading of white spot syndrome virus (WSSV) infections in the black tiger shrimp Penaeus monodon. The test uses 1 sense primer and 3 antisense primers that produce up to 3 PCR products (1100, 526 and 250 base pairs [bp]) depending upon the severity of infection. Specifically, heavy infections (> or = 2 x 10(4) viral particles) of WSSV produce all 3 fragments, while moderate infections (around 2 x 10(3) viral particles) produce 2 (526 and 250 bp) and light infections (20 to 200 viral particles) produce 1 (250 bp). In addition, the technique uses internal control primers that yield a shrimp characteristic fragment for non-infected samples and samples with a low quantity of viral target in order to assure integrity and reproducibility of the PCR assays. The non-stop, single-tube, semi-nested PCR technique is simple and convenient and can detect as little as 5 fg WSSV DNA (20 viral particles) in crude extracts of postlarval samples or extracts of pleopods and haemolymph from larger shrimp.

Yellow head virus from Thailand and gill-associated virus from Australia are closely related but distinct prawn viruses.

Corresponding genomic regions of isolates of yellow head virus (YHV) from Thailand and gill-associated virus (GAV) from Australia were compared by RT-PCR and sequence analysis. PCR primers designed from sequences in the GAV ORF1b polyprotein gene amplified the corresponding 577 nucleotide region of the YHV genome. Comparison of the amplified region indicated 85.1% nucleotide and 95.8% amino acid sequence identity. YHV PCR primers designed to amplify a 135 nucleotide product previously described as a YHV diagnostic probe failed to amplify the corresponding product from GAV RNA. However, the cognate GAV sequence for this and another recently reported YHV sequence were located in an upstream region of the ORF1b gene. A comparison of these sequences indicated identities of 83.0 and 80.9% at the nucleotide level and 86.7 and 86.5% at the amino acid level, respectively. The data indicate that GAV and YHV are closely related but distinct viruses for which differential diagnostic probes can be applied.

Characterization and PCR detection of hepatopancreatic parvovirus (HPV) from Penaeus monodon in Thailand.

Hepatopancreatic parvovirus (HPV) causes disease in several species of penaeid shrimp. Heavy infections may result in poor growth and reduced production for shrimp farmers. From one southern Thai shrimp pond with a high prevalence of HPV infection, 790 shrimp were sampled randomly and the hepatopancreas (HP) removed. Most HP were preserved in liquid nitrogen. However, every 10th HP (79 total) was divided into 2 parts appropriately fixed for examination by transmission electron microscopy (TEM) and light microscopy. Based on light microscopy, the prevalence of HPV infection in the pond was approximately 30% and its presence was confirmed by TEM of parallel samples. The virus was subsequently purified from hepatopancreatic homogenates of the samples preserved in liquid nitrogen. Negative staining of the purified viral preparation revealed unenveloped, icosahedral viral particles 22 to 24 nm in diameter. Agarose gel electrophoresis of nucleic acid extracts revealed the presence of 2 fragments, one very intense (5.8 kb) and the other weak (4.2 kb). The larger fragment was degraded by DNase I and S1 nuclease, indicating single-stranded DNA (ssDNA) characteristic of the viral family Parvoviridae. The smaller fragment was degraded by DNase I but not by S1 nuclease, indicating that it comprised double-stranded DNA. A genomic DNA library of the 5.8 kb ssDNA was constructed in pUC18 and a clone containing a 659 bp fragment specific and sensitive for HPV was selected for sequencing. Based on this sequence, an HPV-specific primer set was designed to yield a 156 bp amplicon by polymerase chain reaction (PCR) amplification. The expected 156 bp amplicon was obtained only in the presence of HPV DNA template (at as little as 1 fg purified DNA) and not with nucleic acid templates extracted from healthy shrimp tissue or other shrimp pathogens. It is hoped that this PCR assay will be useful to shrimp aquaculturists for early detection and screening of shrimp larvae, parental broodstock or other possible carriers of HPV in the shrimp cultivation system.

Experimental transmission of white spot syndrome virus (WSSV) from crabs to shrimp Penaeus monodon.

White spot syndrome virus (WSSV) of the black tiger prawn Penaeus monodon is a recently discovered baculo-like virus disease which is currently the cause of very serious and widespread losses in the shrimp industry in Thailand and elsewhere in Asia. Three suspected crab carriers of this virus commonly found in shrimp-rearing areas were investigated. These were Sesarma sp., Scylla serrata and Uca pugilator. All these crabs could be infected with WSSV by injection and they sustained heavy viral infections for up to 45 d (confirmed by normal histology, specific in situ DNA hybridization and PCR amplification) without visible signs of disease or mortality. All of them also transferred the disease to P. monodon via water while physically separated in aquarium cohabitation tests. Transfer of the virus to the shrimp was monitored using in situ DNA hybridization and PCR assay at 12 h intervals after cohabitation began. With U. pugilator, WSSV could be detected in the shrimp cohabitants after 24 h using PCR amplification and after 60 h using in situ hybridization. With S. serrata, the shrimp were positive for WSSV after 36 h using PCR and after 60 h using DNA in situ hybridization. With Sesarma sp. they were positive after 48 h using PCR and 72 h using in situ hybridization. These laboratory studies demonstrated that crab carriers of WSSV may pose a real threat to cultivated shrimp. However, the studies were carried out in containers with a small volume and with relatively clean sea water as compared to shrimp cultivation ponds. Pond-based studies are now needed to determine whether factors such as pond volume, pond water quality and shrimp and crab behavior can influence the rate and success of transfer.

Genetic structure in wild populations of black tiger shrimp (Penaeus monodon) using randomly amplified polymorphic DNA analysis.

Randomly amplified polymorphic DNA (RAPD) analysis was used to examine genetic variation in wild black tiger shrimp, Penaeus monodon. Specimens were collected from five geographically separated locations (Satun-Trang, Phangnga, and Medan in the Andaman Sea and Chumphon and Trad in the Gulf of Thailand). A total of 100 P. monodon individuals were investigated using seven arbitrarily selected primers. Fifty-eight (72.5%) of eighty reproducible RAPD fragments ranging in size from 200 to 2200 bp were polymorphic. The percentages of polymorphic bands of the five geographic populations investigated varied from 51.5 to 57.7%. The genetic distance between populations and UPGMA dendrograms indicated that the Medan population was genetically different from Thai P. monodon (Dij = 14.976%). Within Thailand, the Satun-Trang P. monodon was separated from the remaining geographic populations with a genetic distance of 2.632%. RAPD analysis in the present study yielded a total of 252 genotypes. A Monte Carlo analysis illustrated geographic heterogeneity in genotype frequencies within this species, suggesting that genetic population structure does exist in this taxon (P < 0.001 for all primers). Signficant differences in genotype frequencies between Thai and Indonesian (Medan) P. monodon were observed (P < 0.0001). Within Thailand, the Andaman Sea P. monodon was significantly different from that of the Gulf of Thailand (P values between 0.0000 and 0.0387), indicating population differentiation between P. monodon from these two main fishery regions of Thailand.

Random amplified polymorphic DNA (RAPD) markers for determination of genetic variation in wild populations of the black tiger prawn (Penaeus monodon) in Thailand.

Random amplified polymorphic DNA (RAPD) analysis was used to amplify the genome of black tiger prawns (Penaeus monodon) to detect DNA markers and assess the utility of the RAPD method for investigating genetic variation in wild P. monodon in Thailand. A total of 200 ten-base primers were screened, and 84 primers yielded amplification products. Six positive primers that gave highly reproducible RAPD patterns were selected for the analysis of three geographically different samples of Thai P. monodon. A total of 70 reproducible RAPD fragments ranging in size from 200 to 2000 bp were scored, and 40 fragments (57%) were polymorphic. The RAPD analysis of broodstocks from three different locales, Satun-Trang, Trat, and Angsila, revealed different levels of genetic variability among the samples. The percentages of polymorphic bands were 48% and 45% in Satun-Trang and Trat, respectively, suggesting a high genetic variability of the two samples to be used in selective breeding programs. Only 25% polymorphic bands were found in the Angsila sample, indicating the lowest polymorphic level among the three samples examined. Primer 428 detected a RAPD marker that was found only in P. monodon originating from Satun-Trang, suggesting the potential use of this marker as a population-specific marker in this species.

A simple, rapid and sensitive detection of salmonella in food by polymerase chain reaction.

A polymerase chain reaction (PCR) method was developed for detection of salmonella in food. A set of PCR primers was designed to amplify a 199 bp salmonella-specific DNA fragment derived from a repetitive DNA of Salmonella Weltevreden. The assay detected all 52 most prevalent serovars found in contaminated food in Thailand and no cross-reaction was observed with other non-salmonella organisms. The limit of detection was 1 fg of purified target DNA or five bacteria from pure culture. The detection of artificially contaminated food performed following a 6 h enrichment step was three bacteria per gram and the result was obtained within 4 h.

Simple nonradioactive DNA hybridization method for identification of sibling species of Anopheles dirus (Diptera: Culicidae) complex.

A simple method was developed for species identification of mosquitoes in the Anopheles dirus complex found in Thailand using horseradish peroxidase-labeled DNA probes and a chemiluminescent detection system. Species-specific DNA probes for Anopheles dirus, species B, C, and D detected 1-5 ng of target DNA, a sensitivity that was comparable with the radioisotopic detection system. Identification of individual mosquitoes was performed by dot-blot analysis of crude mosquito DNA. The method allowed identification using third or fourth instars as well as the adult head, thorax, or abdomen. This technique successfully identified the sibling species of the A. dirus complex in field collections.

DNA fingerprinting and forensic medicine.

In forensic medicine, DNA fingerprinting for identification is becoming a necessary procedure. A method to radiolabel M13 DNA probe by primer extension using a specific oligonucleotide primer was developed. The method specifically labeled the two 15 bp repeats in M13 DNA which hybridize to target DNA giving rise to DNA fingerprinting patterns. The M13 probe labeled by this method has proven useful for individual identification, paternity testing and monitoring reconstitution in bone marrow transplantation. The genetic locus D1S80 and D17S30 containing a variable number of tandem repeats (VNTR) have also been successfully amplified from human genomic DNA isolated from blood (50 ng from each sample) by the polymerase chain reaction (PCR) using oligonucleotide primers complementary to the flanking sequences as primers for amplification. DNA bands were detected by ethidium bromide staining after electrophoresis on agarose gels. Analysis of this VNTR locus was thus achieved without the need for Southern blot or radioactive material. The small size of the DNA fragments produced in the PCR amplification permited good resolution of individual alleles. The precise specification of the number of tandem repeats present in each allelic fragment was reproducible from one analysis to another.

Field evaluation of simplified radioactive and nonradioactive DNA probe methods for the diagnosis of falciparum malaria.

Specific DNA probe hybridization technique is one method of choice for detection of malaria infection. It provides an obvious advantage over conventional microscopy when large numbers of samples are simultaneously monitored. The method was simplified so that preparation and processing of blood specimens were all performed on membrane filters. Background signals generated from blood components were removed by treating samples spotted on the membrane with a series of buffer washes without the necessity of a protease digestion step. Hybridization was monitored using either 32P-or digoxigenin-labeled DNA probe. 849 field samples collected from various malaria endemic areas in Thailand have been evaluated by this protocol and compared with microscopic examination. Sensitivity obtained by this procedure was comparable to that of microscopy at a malaria clinic. The specificities of both types of DNA probes were better than 93%, but digoxigenin-labeled probe performed better than 32P-labeled one when the numbers of parasites were less than 25 per 200 white blood cells.

A highly sensitive, rapid, and simple polymerase chain reaction-based method to detect human malaria (Plasmodium falciparum and Plasmodium vivax) in blood samples.

A highly sensitive, rapid and simple method to detect human malaria in blood samples was developed. Malaria parasite DNA in blood from a fingerprick was directly amplified by the polymerase chain reaction (PCR) using two sets of primers to yield a 206-basepair (bp) product for Plasmodium falciparum and a 183-bp product for P. vivax. Both were easily visualized in an ethidium bromide-stained agarose gel, allowing identification of the two human malaria species in a single amplification reaction. As little as a one P. falciparum and/or P. vivax parasite per microliter of blood was detectable by this method, a sensitivity superior to that of thick blood film microscopy. The total time required for diagnosis of 48 blood samples, starting from fingerprick blood collection, was approximately 4 hr. When compared with microscopic examination by an expert microscopist, results showed a sensitivity of 89% for P. falciparum and 91% for P. vivax and an overall specificity of 94%. Six infected blood samples classified by microscopy as single species were diagnosed by the PCR method as being mixed P. falciparum and P. vivax infections. The high sensitivity, rapidity, and simplicity of the method should make it attractive for a large-scale epidemiology study, follow-up of drug treatment, and immunization trials.

Sensitive detection of Plasmodium falciparum in blood and mosquito by DNA amplification.

By means of enzymatic amplification of Plasmodium falciparum DNA using the polymerase chain reaction (PCR), we have been able to detect as little as 20 parasites in 20 ml infected human blood based on the visualization of a 206 bp fragment in ethidium bromide-stained agarose gel. Comparison, through microscopic examination of 2030 blood samples collected from various endemic areas in Thailand, indicates that the PCR-based detection system is 96% specific and 81% sensitive, with a disagreement of 6.6%. The same protocol can be applied to detect a minimum of 10 sporozoites or a single oocyst dissected from P. falciparum-infected mosquito.

Polymerase chain reaction detection of Plasmodium falciparum in mosquitoes.

The polymerase chain reaction (PCR) procedure using a primer set derived from a repetitive deoxyribonucleic acid (DNA) sequence specific to Plasmodium falciparum was used to detect parasite DNA in mosquitoes. In laboratory-infected mosquitoes, PCR could detect as few as 10 sporozoites in a dissected salivary gland and a single oocyst in a dissected midgut. The ability to detect P. falciparum DNA in wild-caught mosquitoes indicated an advantage of the PCR over enzyme-linked immunosorbent assay for the detection of Plasmodium sporozoites in mosquitoes with low-grade parasite infections.

A specific DNA probe which identifies Babesia bovis in whole blood.

A genomic library of Babesia bovis DNA from the Mexican strain M was constructed in plasmid pUN121 and cloned in Escherichia coli. Several recombinants which hybridized strongly to radioactively labeled B. bovis genomic DNA in an in situ screening were selected and further analyzed for those which specifically hybridized to B. bovis DNA. It was found that pMU-B1 had the highest sensitivity, detecting 25 pg of purified B. bovis DNA, and 300 parasites in 10 microliters of whole infected blood, or 0.00025% parasitemia. pMU-B1 contained a 6.0 kb B. bovis DNA insert which did not cross-hybridize to Babesia bigemina, Trypanosoma evansi, Plasmodium falciparum, Anaplasma marginale, Boophilus microplus and cow DNA. In the Southern blot analysis of genomic DNA, pMU-B1 could differentiate between two B. bovis geographic isolates, Mexican strain M and Thai isolate TS4. Thus, the pMU-B1 probe will be useful in the diagnosis of Babesia infection in cattle and ticks, and in the differentiation of B. bovis strains.

Improved resolution and sensitivity of human DNA fingerprinting by specific-primed labelling of M13 DNA.

A method to label M13 DNA probe by primer extension using a specific oligonucleotide primer is described. The method specifically labels the two 15-bp repeats in M13 DNA which hybridize to target DNA giving rise to DNA fingerprinting patterns. The M13 probe labelled by this method gave superior DNA fingerprinting patterns that that labelled by random primers. As little as 0.25 microgram of target DNA was sufficient for DNA fingerprinting. Non-isotopic labelling by the specific primer also showed improved DNA fingerprinting pattern. The results demonstrate the methodology to improve DNA fingerprinting based on M13 DNA probe.

A novel detection of a single Plasmodium falciparum in infected blood.

Detection of Plasmodium falciparum malaria by a specific DNA probe is a highly promising means for epidemiological surveillance of human malaria. However, none of presently available DNA probe methods could detect as little as a few parasites in infected blood. By amplification of a specific 206 base pairs P. falciparum DNA sequence using the polymerase chain reaction (PCR), as little as 0.01 picogram DNA or one-half of a parasite was sufficient for a specific detection. A PCR procedure for detection of P. falciparum in infected blood without prior DNA extraction was also developed which was sensitive for a single parasite. The procedure was simple and should be applicable for a large scale epidemiological study involving a very low parasitemia situation.

Detection of Plasmodium falciparum using a cloned DNA probe: a simple procedure suitable for field application.

A simple procedure was developed for spotting blood samples directly onto nylon membrane filter, without the necessity to treat samples with pronase or proteinase K, followed by hybridizing with 32P-labelled DNA probe, pUNK1-45. This probe detected specifically P. falciparum DNA and did not cross react with DNA from man, P. knowlesi, P. chabaudi or P. cynomolgi. The probe was sensitive to detect a parasitemia of 0.001% in 20 microliters of blood.

Molecular structure of human seminal coagulum: the role of proteolysis.

The techniques of SDS-polyacrylamide slab gel electrophoresis and a sensitive fluorescamine-based method were used to study autoproteolysis of human seminal coagulum at various pH. The major protein bands of 72 and 55 Kd were optimally degraded at pH 7.5. Degradation at pH 3.5 was preceded by a lag period of 30 min. When the seminal coagulum was incubated for 5 min at pH 7.5 and then adjusted to pH 3.5, degradation was more rapid and complete than at pH 3.5 only. Autoproteolytic degradation product of discreted sizes were obtained when compared with degradation in the presence of pronase.

Molecular structure of human seminal coagulum: the role of disulfide bonds.

Using sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis in the presence of 100 mM 2-mercaptoethanol, freshly ejaculated human semen showed two major protein bands of 72 and 55 kd. During liquefaction these two protein bands were degraded to low molecular weight proteins of 10-15 kd. In the absence of 2-mercaptoethanol, larger proteins such as those of 243, 221 and 195 kd were obtained which could be converted to 72 and 55 kd proteins by the action of the sulfhydryl reducing agent. Liquefaction was enhanced in the presence of 2-mercaptoethanol. These results indicated that one of the factors involved in the formation of human seminal coagulum is disulfide linkage between proteins of 72 and 55 kd.