High juvenile hormone titre and abdominal activation of JH signalling may induce reproduction of termite neotenics.

Termite castes are a key example of polyphenism, in which reproductive division of labour is clearly seen in colonies. The reproductive castes in termites include primary and neotenic reproductives; primary reproductives found a new colony whereas neotenics succeed them in the reproductive role when the primary reproductives die or become senescent. Neotenics usually differentiate from nymphs or workers by developing functional gonads while retaining juvenile characteristics; however, the developmental mechanism during neotenic differentiation remains poorly understood. Juvenile hormone (JH) mediates a number of aspects of developmental regulation in caste differentiation in termites. In the present study we quantified JH titres in neotenic reproductives of Reticulitermes speratus, and compared these with other developmental stages. In addition, expression changes in JH signalling gene homologues (Methoprene-tolerant [Met], Krüppel-homolog1, Broad-Complex) in the head, thorax and abdomen were investigated during neotenic differentiation. Finally, we examined the function of Met in reproduction of neotenics by RNA interference (RNAi). Our results showed that the JH titres of neotenics were significantly higher than those of nymphs and workers. JH signalling genes were highly expressed in neotenic abdomens, compared with those in workers and nymphs. Met RNAi resulted in the inhibition of vitellogenin gene expression in newly moulted neotenics. These results suggest that the fertility of neotenics might be controlled by a large increase of JH titres and body-part-specific activation of JH signalling pathways.

Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.

Two new methods were used to establish a rapid and highly sensitive prenatal diagnostic test for sickle cell anemia. The first involves the primer-mediated enzymatic amplification of specific beta-globin target sequences in genomic DNA, resulting in the exponential increase (220,000 times) of target DNA copies. In the second technique, the presence of the beta A and beta S alleles is determined by restriction endonuclease digestion of an end-labeled oligonucleotide probe hybridized in solution to the amplified beta-globin sequences. The beta-globin genotype can be determined in less than 1 day on samples containing significantly less than 1 microgram of genomic DNA.

Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase.

A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme, isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified. Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule present only once in a sample of 10(5) cells.

Detection of ras gene mutations in pancreatic juice and peripheral blood of patients with pancreatic adenocarcinoma.

Pancreatic adenocarcinomas are known to have a high incidence of K-ras gene mutations. Differential diagnosis of pancreatic cancer and chronic pancreatitis sometimes presents a clinical dilemma. We recently developed a highly sensitive and specific polymerase chain reaction capable of detecting 3-30 copies of mutant K-ras genes harboring codon 12 single base changes in the presence of 300,000 normal copies. Mutant ras genes were detected in DNA purified from pancreatic juice from all 6 cases of pancreatic adenocarcinoma and 1 case of intraductal papillary neoplasms of the pancreas. In 2 of 6 other cases with pancreatic adenocarcinoma, circulating metastatic cells were detected in DNA purified from peripheral blood. Activated ras genes were not found in pancreatic juice of three control cases (chronic pancreatitis and choledocholithiasis) or in the peripheral blood of two patients with insulinomas. Notable conclusions of this study are that there can be significant levels of shed tumor cells in peripheral blood and an even higher number in pancreatic juice. In addition, two different K-ras mutations were found in some patients.

Genetic analysis using polymerase chain reaction-amplified DNA and immobilized oligonucleotide probes: reverse dot-blot typing.

The reverse dot-blot method is a simple and rapid diagnostic procedure that allows screening of sample for a variety of mutations/polymorphisms in a single hybridization reaction. Several methods of immobilizing the oligonucleotide probes are discussed. The reverse dot-blot method has several unique properties that are valuable in a diagnostic setting: (1) the typing results from a single sample can be located on a single strip. This facilitates scanning and interpretation of the probe reactivity patterns and minimizes the potential for user error. (2) The test can utilize premade typing strips. This minimizes user labor as well as error potential and allows the use of standardized reagents. (3) Unlike dot-blot/oligonucleotide typing, only the PCR product is labeled, eliminating the potential problem of probes labeled to different specific activities. This method has already been used in the areas of forensic genetic typing (the HLA-DQ alpha Amplitype test), tissue typing for transplantation (the HLA-DR beta) test, cystic fibrosis screening, as well as in a variety of research applications.

Analysis of 16 cystic fibrosis mutations in Mexican patients.

We carried out molecular analysis of 80 chromosomes from 40 unrelated Mexican patients with a diagnosis of cystic fibrosis. The study was performed in two PCR steps: a preliminary one to identify mutation delta F508, the most frequent cause of cystic fibrosis worldwide, and the second a reverse dot-blot with allele-specific oligonucleotide probes to detect 15 additional common mutations in the Caucasian population. A frequency of 45% for delta F508 was found, making it the most common in our sample of Mexican patients. Another five mutations (G542X, 3,849 + 10 kb C-->T, N1303K, SN549N, and 621 + 1 G-->T) were detected, and those accounted for 11.25%. The remaining mutations (43.75%) were undetectable with the methodology used.

Body weight change and nitrogen efficiencies in growing and adult rats fed diets containing various proportions of essential amino acids to total amino acids.

By using amino acid mixtures, a comparative study of nutritional effects of dietary essential amino acid proportions (EA %) has been made between growing and adult rats. Included were adult rats which were repleting from an 8-day protein depletion. It was shown that for growing rats at least 55 EA% was required to attain the maximum values of growth, nitrogen balance, nitrogen balance efficiency (nitrogen balance/nitrogen intake) and protein efficiency ratio (PER). The maximum biological value was found to be 94 at the 50 EA% level. For adult rats, at least 40 EA% was required to gain the maximum values of nitrogen balance and nitrogen balance efficiency under both maintenance and repletion. The biological values were found to be nearly 60 and 80 for maintenance and repletion, respectively.

A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions.

Specific, end-labeled DNA fragments can be simply and rapidly prepared using the polymerase chain reaction (PCR). Such fragments are suitable for use in DNase I protection footprint assays, chemical sequencing reactions, and for the production and analysis of paused RNA polymerase transcription complexes. Moreover, a general means of introducing a specific mutation at any position along the length of such PCR-generated fragments is described. These procedures, which can circumvent the need for large-scale phage or plasmid growths, preparative gel-electrophoresis and the screening of molecular clones, can facilitate the rapid study of sequence-specific interactions of proteins and DNA. A rapid means of removing excess oligonucleotide primers from completed PCRs is also described.

Analysis by molecular cloning of the human class II genes.

The HLA class II genes control immune responsiveness to defined antigens; they encode cell surface heterodimers composed of alpha and beta glycopeptides. Recently, cDNA and genomic clones encoding these chains have been isolated, which allows molecular analysis of the class II genes. cDNA clones encoding the alpha chain of the HLA-DR antigen as well as that of another HLA class II antigen have been identified and characterized by nucleotide sequence analysis. These clones have been used as probes to isolate additional class II alpha cDNA clones in cDNA libraries and to identify polymorphisms in genomic DNA. Polymorphic restriction sites have been localized within the HLA-DR alpha gene and used as genetic markers in the analysis of families and of disease (insulin-dependent diabetes mellitus) and control populations. In addition, cDNA clones encoding the DR beta and DC beta chains were used as hybridization probes to identify DNA polymorphism. cDNA clones encoding the DR gamma (Ii) chain have also been identified; unlike the DR alpha and DR beta loci, the DR gamma gene is located on some chromosome other than chromosome 6. The genetic complexity of the human class II alpha and beta loci, as revealed by analysis with cDNA and genomic clones, is greater than that of the murine class II genes. The extent of that complexity will be defined by future work in this area.

Analysis of DNA extracted from formalin-fixed, paraffin-embedded tissues by enzymatic amplification and hybridization with sequence-specific oligonucleotides.

The "polymerase chain reaction" (PCR) procedure for amplifying specific gene sequences has recently been combined with sequence-specific oligonucleotide (SSO) probe hybridization to develop a highly sensitive, rapid, and simple method for analyzing allelic variations in genomic DNA. In the present study we have used PCR/SSO to analyze partially purified DNA extracted from formalin-fixed, paraffin-embedded tissue specimens. We report that this DNA, including samples that were partially degraded, proved to be suitable for analysis by the PCR/SSO procedure.

Analysis of RAS gene mutations in acute myeloid leukemia by polymerase chain reaction and oligonucleotide probes.

In vitro DNA amplification followed by oligonucleotide dot blot analysis were used to study RAS gene mutations in acute myeloid leukemia (AML). Fifty-two presentation AML DNAs were screened for mutations in codons 12, 13, and 61 of NRAS and in codons 12 and 61 of KRAS and HRAS. Fourteen (27%) contained mutations--all in NRAS and predominantly in codon 12. The most common amino acid substitution identified was of glycine by aspartic acid at codon 12 (7/18), with a G----A transition being the most common base change (11/18). No particular correlation was observed between disease subtype and the incidence or type of NRAS mutation. In DNA samples from four patients, 2 NRAS mutations were found to coexist. NIH 3T3 focus-formation assays revealed that in each case the mutations were present in different NRAS alleles. We also report the absence of a mutated RAS gene in relapse DNAs of four patients in which a RAS oncogene had been detected at presentation. These observations suggest that RAS mutations arise as part of the evolution of neoplastic transformation.

Reverse dot-blot detection of Thai beta-thalassaemia mutations.

Pending curative therapy, newborn screening and prenatal diagnosis are essential to the management of beta thalassaemia. Diagnosis using electrophoretic methods is difficult in the presence of composite phenotypes and high Hb F levels. Direct DNA detection of mutant alleles circumvents both problems, but the enormous diversity of beta-thalassaemia mutations poses challenges for this approach. Among PCR-based tests, the reverse dot-blot method enables screening several mutations with a single hybridization reaction. Unfortunately it has often been targeted to only the common mutations of a particular ethnic population, necessitating the use of more arduous detection methods for the less common mutations. We developed a reverse dot-blot strip for the 10 beta-thalassaemia mutations, including the beta-thalassaemic haemoglobinopathies Hb E and Hb Malay, that account for 96% of beta thalassaemia in Thailand, and another strip for six less common Thai mutations. The second strip precludes the need for more technically challenging methods. To avoid problems associated with secondary structure of amplified full-length target DNA, we amplified and labelled beta-globin DNA as two shorter fragments that encompassed all known Thai mutations. Reverse dot-blotting is a rapid, accurate method for detecting beta-thalassaemia mutations.

T-DNA from Agrobacterium Ti plasmid is in the nuclear DNA fraction of crown gall tumor cells.

The crown gall teratoma tumor line BT37, incited by Agrobacterium tumefaciens strain T37, has been found to contain part of the tumor-inducing plasmid, pTi T37, of the inciting strain. This foreign DNA segment, called T-DNA, is maintained at several copies per diploid tumor cell. We have examined subcellular DNA fractions from this tumor line in an effort to determine whether T-DNA is in chloroplasts, mitochondria, or nuclei. Tumor cell chloroplast DNA exhibited EcoRI and Bst I endonuclease cleavage patterns identical to those of normal tobacco chloroplast DNA. Tumor cell mitochondrial DNA exhibited a complex Bst I cleavage pattern that did not differ detectably from that of normal tobacco mitochondrial DNA. Southern blots of tumor chloroplast and mitochondrial cleavage products did not hybridize with labeled pTi T37 DNA, whereas blots of tumor cell nuclear DNA cleavage products hybridized strongly. We conclude that T-DNA is located not in chloroplasts or mitochondria but rather in the nuclear fraction of this tumor line.

T-DNA of a crown gall teratoma is covalently joined to host plant DNA.

Agrobacterium tumefaciens strains containing tumour-inducing (Ti) plasmids incite cancerous growths called crown galls when inoculated into wounded dicotyledonous plants. Tumour tissue can be cultured axenically in vitro, and exhibits a transformed phenotype in the absence of the inciting bacterium. Transformed cells grow autonomously, are auxin and cytokinin autotrophic in vitro and synthesize opines, novel amino acid derivatives dictated by Ti plasmid genetic information. A small segment of the Ti plasmid, termed T-DNA is maintained in axenic tumour cells. Mitochondrial and chloroplast DNA from a crown gall teratoma are free from T-DNA, whereas nuclear DNA contains T-DNA in amounts similar to that in total tumour cell DNA. T-DNA appears to be attached to what is presumably plant DNA in the crown gall tumour cell: Southern blot analysis of tumour DNA digested with restriction endonucleases reveals T-DNA fragments that are not fully homologous to Ti plasmid DNA. We report here the isolation by molecular cloning of a 'border fragment' T-DNA and flanking plant DNA from the crown gall teratoma BT37 and show that T-DNA is covalently joined to a repeated DNA element of the tobacco nuclear genome.

Mapping of the genes encoding the HLA-DR alpha chain and the HLA-related antigens to a chromosome 6 deletion by using genomic blotting.

We have used genomic blotting with DNA from a human cell line that has a small deletion on chromosome 6 (6.3.6) and from its parent cell line (T5-1) to map DNA fragments complementary to cloned DNA sequences encoding the HLA-B7 antigen (class I) and the alpha chain of the HLA-DR antigen (class II). The 6.3.6 variant fails to express the HLA-A, -B, -C, and -DR and MB specificities associated with one of the parental T5-1 haplotypes and has a visible deletion in the short arm of one chromosome 6 (1). The gene locus assignment was based on the expectation that, if the chromosomal location of the DNA sequences used as a hybridization probe were within the deletion, then the relative amount or size (or both) of genomic restriction fragments that hybridize to the probe in T5-1 and in 6.3.6 DNAs should differ predictably. By comparing the genomic blot patterns from T5-1 and 6.3.6 DNAs, we have shown directly that the loss of haplotype expression was due to deletion of the structural genes and have mapped the structural gene for the HLA-DR alpha chain to the chromosomal location (6p2105-6p23) defined by the 6.3.6 deletion. A cDNA clone encoding the alpha chain of the HLA-DR antigen hybridized to two genomic fragments, 4.2 and 3.8 kilobases long, generated by Bgl II digestion of T5-1 DNA. The 4.2-kilobase fragment was absent from DNA derived from the 6.3.6 deletion variant. Thus, this fragment could be assigned to the parental chromosome 6 with the A1, B8, DR3 haplotype, and the 3.8-kilobase fragment, to the chromosome 6 with the A2, B27, DR1 haplotype. In addition, comparison of the T5-1 and 6.3.6 genomic blot patterns obtained with the HLA-B7 probe revealed dosage differences for all of the class I genomic fragments generated by BamHI digestion, suggesting that all of the class I loci map to the region 6p2105-6p23.

Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes.

The analysis of DNA for the presence of particular mutations or polymorphisms can be readily accomplished by differential hybridization with sequence-specific oligonucleotide probes. The in vitro DNA amplification technique, the polymerase chain reaction (PCR), has facilitated the use of these probes by greatly increasing the number of copies of target DNA in the sample prior to hybridization. In a conventional assay with immobilized PCR product and labeled oligonucleotide probes, each probe requires a separate hybridization. Here we describe a method by which one can simultaneously screen a sample for all known allelic variants at an amplified locus. In this format, the oligonucleotides are given homopolymer tails with terminal deoxyribonucleotidyltransferase, spotted onto a nylon membrane, and covalently bound by UV irradiation. Due to their long length, the tails are preferentially bound to the nylon, leaving the oligonucleotide probe free to hybridize. The target segment of the DNA sample to be tested is PCR-amplified with biotinylated primers and then hybridized to the membrane containing the immobilized oligonucleotides under stringent conditions. Hybridization is detected nonradioactively by binding of streptavidin-horseradish peroxidase to the biotinylated DNA, followed by a simple colorimetric reaction. This technique has been applied to HLA-DQA genotyping (six types) and to the detection of Mediterranean beta-thalassemia mutations (nine alleles).

Phenotypes of fission yeast defective in ubiquinone production due to disruption of the gene for p-hydroxybenzoate polyprenyl diphosphate transferase.

Ubiquinone is an essential component of the electron transfer system in both prokaryotes and eukaryotes and is synthesized from chorismate and polyprenyl diphosphate by eight steps. p-Hydroxybenzoate (PHB) polyprenyl diphosphate transferase catalyzes the condensation of PHB and polyprenyl diphosphate in ubiquinone biosynthesis. We isolated the gene (designated ppt1) encoding PHB polyprenyl diphosphate transferase from Schizosaccharomyces pombe and constructed a strain with a disrupted ppt1 gene. This strain could not grow on minimal medium supplemented with glucose. Expression of COQ2 from Saccharomyces cerevisiae in the defective S. pombe strain restored growth and enabled the cells to produce ubiquinone-10, indicating that COQ2 and ppt1 are functional homologs. The ppt1-deficient strain required supplementation with antioxidants, such as cysteine, glutathione, and alpha-tocopherol, to grow on minimal medium. This suggests that ubiquinone can act as an antioxidant, a premise supported by our observation that the ppt1-deficient strain is sensitive to H(2)O(2) and Cu(2+). Interestingly, we also found that the ppt1-deficient strain produced a significant amount of H(2)S, which suggests that oxidation of sulfide by ubiquinone may be an important pathway for sulfur metabolism in S. pombe. Ppt1-green fluorescent protein fusion proteins localized to the mitochondria, indicating that ubiquinone biosynthesis occurs in the mitochondria in S. pombe. Thus, analysis of the phenotypes of S. pombe strains deficient in ubiquinone production clearly demonstrates that ubiquinone has multiple functions in the cell apart from being an integral component of the electron transfer system.