Modified nucleosides and the chromatographic and aminoacylation behavior of tRNA(Ile) from Escherichia coli C6.

Transfer RNA from Escherichia coli C6, a Met-, Cys-, relA- mutant, was previously shown to contain an altered tRNA(Ile) which accumulates during cysteine starvation (Harris, C.L., Lui, L., Sakallah, S. and DeVore, R. (1983) J. Biol. Chem. 258, 7676-7683). We now report the purification of this altered tRNA(Ile) and a comparison of its aminoacylation and chromatographic behavior and modified nucleoside content to that of tRNA(Ile) purified from cells of the same strain grown in the presence of cysteine. Sulfur-deficient tRNA(Ile) (from cysteine-starved cells) was found to have a 5-fold increased Vmax in aminoacylation compared to the normal isoacceptor. However, rates or extents of transfer of isoleucine from the [isoleucyl approximately AMP.Ile-tRNA synthetase] complex were identical with these two tRNAs. Nitrocellulose binding studies suggested that the sulfur-deficient tRNA(Ile) bound more efficiently to its synthetase compared to normal tRNA(Ile). Modified nucleoside analysis showed that these tRNAs contained identical amounts of all modified bases except for dihydrouridine and 4-thiouridine. Normal tRNA(Ile) contains 1 mol 4-thiouridine and dihydrouridine per mol tRNA, while cysteine-starved tRNA(Ile) contains 2 mol dihydrouridine per mol tRNA and is devoid of 4-thiouridine. Several lines of evidence are presented which show that 4-thiouridine can be removed or lost from normal tRNA(Ile) without a change in aminoacylation properties. Further, tRNA isolated from E. coli C6 grown with glutathione instead of cysteine has a normal content of 4-thiouridine, but its tRNA(Ile) has an increased rate of aminoacylation. We conclude that the low content of dihydrouridine in tRNA(Ile) from E. coli cells grown in cysteine-containing medium is most likely responsible for the slow aminoacylation kinetics observed with this tRNA. The possibility that specific dihydrouridine residues in this tRNA might be necessary in establishing the correct conformation of tRNA(Ile) for aminoacylation is discussed.

Isolation and characterization of the tandemly repeated U6 genes from the sea urchin Strongylocentrotus purpuratus.

The tandemly repeated U6 genes were isolated from the sea urchin Strongylocentrotus purpuratus. Each 1.8 kb repeat unit contains a single U6 RNA sequence. There are no sequence similarities between the U6 promoter and other sea urchin snRNA genes, other than a long polypyrimidine tract 3' of the U6 sequence.

Molecular diagnostics of infectious diseases: state of the technology.

In this review, the basic technologies and procedures currently used in clinical laboratories performing molecular diagnostics are described. Special emphasis on specimen processing has been made since it is one of the most challenging steps involved in molecular testing. Representative examples are given for each type of technology, especially tests that are currently available in the market. The types of hybridization-based and amplification-based procedures are detailed. Finally, current problems and future developments are discussed.

Enzymatic and molecular diagnosis of Gaucher disease.

Advances in the knowledge of the molecular genetics of Gaucher disease has made diagnosis more certain. Carrier detection in kindreds in which the responsible mutation has been defined is completely reliable now. Coupled with enzymatic assays, the diagnostic capabilities are greater than before. Use of these methods provides important information to individuals at risk and allows them to make critical decisions. The new, simplified methods reviewed in this article permit the molecular diagnosis of the disease and carrier stage of large numbers of samples within 1 week.

DNA fingerprinting of crude bacterial lysates using degenerate RAPD primers.

Methods for identifying isolates of various pathogenic bacteria by DNA fingerprinting with random primers (RAPD) have been described recently. In these methods many primers are screened and the primers that generate the most informative DNA pattern are selected. A new strategy that simplifies the primer selection process for RAPD fingerprinting has been developed in our laboratory. In this approach, one or more degenerate nucleotides is introduced into the core RAPD primer sequence at various nucleotide positions. Results show that a single degenerate nucleotide in the primer sequence can significantly change the DNA profile obtained for the same template. The more removed the degenerate nucleotide is from the 3' end of the primer, the less dramatic is its effect on banding pattern. This method utilizing degenerate RAPD (D-RAPD) primers was tested on clinical isolates of Legionella pneumoniae, and results were confirmed with nondegenerate RAPD primers. Results obtained with D-RAPD primers were in total agreement with those obtained with nondegenerate RAPD primers. We propose that the use of a core RAPD primer sequence with one or more degenerate nucleotide(s) at various positions can expedite the generation of unique DNA fingerprints individual organisms. A general method for selecting the most useful fingerprinting RAPD primers is discussed.

Cysteine starvation, isoleucyl-tRNAIle, and the regulation of the ilvGEDA operon of Escherichia coli.

The involvement of undermodified tRNA in the regulation of the ilvGEDA operon has been investigated using Escherichia coli C6, a relA-, Cys-, Met- mutant. This strain accumulates thionucleotide-deficient or methyl-deficient tRNA when starved for cysteine or methionine, respectively. The levels of threonine deaminase, the ilvA gene product, and transaminase B, the ilvE gene product, were both lower in cysteine-starved cells, as compared with either growing or methionine-starved cultures. When cysteine was added to cysteine-starved cells, growth ensued promptly and both enzyme activities returned to control levels. Treatment of recovering cultures with valine limited growth by isoleucine limitation, but did not cause a derepression of the ilvGEDA operon. Valine treatment of nonstarved or methionine-starved cells led to the expected increase in threonine deaminase and transaminase B activities. Cysteine-starved cells slowly regained the ability to derepress the operon after 3 h of recovery in complete medium. In contrast, the induction of the lac operon was normal in cysteine-starved cultures, even in the presence of valine. The loss of derepressibility of the ilvGEDA operon was correlated with the presence of a kinetically and chromatographically altered tRNAIle in cysteine-starved cells. No changes in tRNAIle were observed after methionine starvation. Using the periodate method, we found that the charging of tRNAIle increased from the normal level of 60 to 80% or greater after starvation for cysteine. Under conditions where the ilvGEDA operon was fully derepressed in nonstarved cells, the charging of tRNAIle fell to 27%. Unexpectedly, nearly identical results were obtained with cysteine-starved cells after an identical derepression test. These results suggest that factors other than the aminoacylation state of tRNAIle may be important in the regulation of this operon. In particular, modifications to tRNA which involve cysteine may be necessary for controlling the expression of the ilvGEDA operon in E. coli.

A developmental switch in sea urchin U1 RNA.

The sequence of U1 RNA has been determined in the eggs and embryos of two sea urchins, Lytechinus variegatus and Strongylocentrotus purpuratus. In both species the sequence of the U1 RNA changes as the embryos progress through development. The sequence of the major U1 RNA in the eggs of the two species differs in two nucleotides, while the sequence of the U1 RNA present in the late embryos and somatic tissue is identical in the two species. The U1 RNA in eggs and early embryos is primarily derived from the tandemly repeated gene set, which is not expressed in somatic tissues.

A new diagnostic test for Gaucher disease suitable for population screening.

A new test for the diagnosis of Gaucher disease is described. The test is designed to screen large numbers of clinical specimens from high-risk populations. It consists of duplex PCR amplification of genomic DNA followed by hybridization to alkaline phosphatase-conjugated allele-specific oligonucleotide probes (ASOs). High melting temperature PCR primers were used to increase specificity and eliminate the need for a separate annealing step. All hybridization and washing steps were performed at one temperature. Chemiluminescent detection of signals is fast, and results are easily interpreted directly from x-ray films. Currently, the test is being used in our laboratories to screen Ashkenazi Jewish populations in whom Gaucher disease is common.

Isolation and characterization of developmentally regulated sea urchin U2 snRNA genes.

Genes encoding the U2 snRNA have been isolated from the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. Representatives of tandemly repeated gene sets have been isolated from both sea urchin species and a unique U2 gene has also been isolated from L. variegatus. The sequence of the U2 snRNA encoded by the tandemly repeated genes differs in two nucleotides between S. purpuratus and L. variegatus. The unique U2 gene from L. variegatus encodes the same U2 RNA as the tandemly repeated genes. There is a change in the U2 genes expressed between morula and pluteus embryos as judged by a change in the U2 RNA sequence in S. purpuratus embryos. The tandemly repeated genes were expressed at a higher rate in blastula than in gastrula stage relative to the single-copy gene, when the two genes were injected into sea urchin zygotes.

The U1 snRNA gene repeat from the sea urchin (Strongylocentrotus purpuratus): the 70 kilobase tandem repeat ends directly 3' to a U1 gene.

Lambda phage clones containing multiple copies of the 1.1 kb tandemly repeated unit of the sea urchin (S. purpuratus) U1 RNA genes were isolated from a gene library. The 1.1 kb repeat unit encodes a single copy of the predominant U1 RNA expressed in oocytes and embryos prior to the blastula stage. The tandem repeat unit is about 80 kb in size and is probably present one time per haploid genome as judged by pulsed-field electrophoresis of sperm DNA digested with restriction enzymes which do not cut in the repeat unit. Two of the phage contained DNA flanking the repeat unit as well as several repeat units. The tandem repeat unit ends just 3' to the U1 coding region. There is only limited homology in the 5' flanking region with U1 snRNA genes from the sea urchin L. variegatus.

Typing of Legionella pneumophila serogroup 1 isolates by degenerate (D-)RAPD fingerprinting.

A new method to identify clonal strains of pathogenic bacteria has been developed recently in this laboratory. The method utilizes degenerate random amplified polymorphic DNA primers (D-RAPD) to amplify random fragments in crude bacterial lysates, generating reproducible DNA banding profiles or fingerprints. We use this method to type outbreak and non-outbreak isolates of Legionella pneumophila serogroup 1 from four hospitals near to, and affiliated with the University of Pittsburgh Medical Center. Patient isolates from a large outbreak, and nearly half of the contemporaneous environmental isolates showed the same DNA profile. Other isolates derived from non-outbreak patients showed easily distinguishable profiles. Other Legionella isolates collected between 1984 and 1994 were also analysed by this method. Our studies demonstrate that four strains were common among patient and environmental isolates at the four hospitals. These strains were also found to be different from a limited number of isolates from outside the Pittsburgh area. Because of its speed, simplicity and powerful discriminating ability, we believe that the D-RAPD approach provides epidemiologists and hospital infection control teams with a powerful tool in their efforts in analysing and terminating infection outbreaks.

Gaucher disease: studies of phenotype, molecular diagnosis and treatment.

This report summarizes the results on 39 patients with Gaucher disease who have been genotyped, evaluated, and/or followed at this center. Mutation analysis for 4 common mutations; N370S, L444P, 84gg and IVS2 (+1), was performed for all patients. Mutation analysis identified both mutant alleles in 69% and at least one mutant allele in 90% of all chromosomes. This study group of 39 patients included 32 type 1, four type 2 and three type 3 patients. We include the details of the clinical course of two patients with Gaucher disease treated with enzyme replacement therapy (ERT). One patient with chronic neuronopathic Gaucher disease has been treated with enzyme replacement therapy (ERT) at a dose of 60 U/kg every 2 weeks since 2.5 years of age and has shown no progression of neurologic involvement. A second patient with non-neuronopathic Gaucher disease has demonstrated an unusually delayed response to ERT. No clinical response was noted following 17 months of treatment at 60 U/kg every 2 weeks. Only after the dose was increased to 60 U/kg every week was a clinical response evident. Response to treatment at 15 U/kg every 2 weeks was variable in the four type 1 patients treated at the lower dose. In two of these patients with identical genotypes, one patient demonstrated a positive clinical response to low dose treatment while the other patient did not.

Loss of heterozygosity in spontaneous and chemically induced tumors of the B6C3F1 mouse.

The B6C3F1 mouse is used worldwide to gauge the carcinogenic hazard posed by chemicals to humans. An assessment of the ability of this rodent model to predict human neoplasia requires an evaluation of similarities and differences in the genetics of tumor formation between these two species. We examined 142 spontaneous and chemically-induced liver tumors isolated from the B6C3F1 mouse for losses of heterozygosity (LOH) at 78 polymorphic loci and compared these results to genetic changes known to occur in human hepatocellular carcinoma. Approximately a third of the 142 mouse tumors exhibited LOH, suggesting that tumor suppressor gene inactivation may be involved in the formation of mouse liver tumors. Most of the LOH observed was restricted to seven chromosome sites and most of the tumors that underwent LOH lost alleles from only one of those seven sites. The relatively few losses seen in these mouse tumors distinguished them from clinical stage human tumors in that, in the mouse tumors, interstitial deletions appeared more frequently than losses of whole chromosomes. Only four mouse tumors lost a whole chromosome. LOH occurred at loci of the mouse genome syntenic to areas of the human genome known to harbor the Wilms', retinoblastoma, APC, MCC and DCC tumor suppressor genes; these genes have never been associated with hepatocellular carcinomas. Losses observed on chromosomes 5 and 8 (syntenic to human chromosomes 4 and 16) suggest tumor suppressor genes that are common to hepatocellular carcinomas from both species, while losses on chromosome 9 suggest involvement of a previously unidentified tumor suppressor gene.

Molecular biology of glucocerebrosidase and the treatment of Gaucher disease.

The inherited deficiency of glucocerebrosidase results in a group of sphingolipid storage disorders referred to collectively as Gaucher disease. Study of the biochemistry and cell biology of glucocerebrosidase has made possible an effective enzyme replacement therapy for the disease. Definition of the molecular genetics of glucocerebrosidase has improved diagnostic capabilities and presents the exciting possibility of a cure through gene therapy.

Distribution and evolution of CTG repeats at the myotonin protein kinase gene in human populations.

We have analyzed the CTG repeat length and the neighboring Alu insertion/deletion (+/-) polymorphism in DNA samples from 16 ethnically and geographically diverse human populations to understand the evolutionary dynamics of the myotonic dystrophy-associated CTG repeat. Our results show that the CTG repeat length is variable in human populations. Although the (CTG)5 repeat is the most common allele in the majority of populations, this allele is absent among Costa Ricans and New Guinea highlanders. We have detected a (CTG)4 repeat allele, the smallest CTG known allele, in an American Samoan individual. (CTG) > or = 19 alleles are the most frequent in Europeans followed by the populations of Asian origin and are absent or rare in Africans. To understand the evolution of CTG repeats, we have used haplotype data from the CTG repeat and Alu(+/-) locus. Our results are consistent with previous studies, which show that among individuals of Caucasian and Japanese origin, the association of the Alu(+) allele with CTG repeats of 5 and > or = 19 is complete, whereas the Alu(-) allele is associated with (CTG)11-16 repeats. However, these associations are not exclusive in non-Caucasian populations. Most significantly, we have detected the (CTG)5 repeat allele on an Alu(-) background in several populations including Native Africans. As no (CTG)5 repeat allele on an Alu(-) background was observed thus far, it was proposed that the Alu(-) allele arose on a (CTG)11-13 background. Our data now suggest that the most parsimonious evolutionary model is (1) (CTG)5-Alu(+) is the ancestral haplotype; (2) (CTG)5-Alu(-) arose from a (CTG)5-Alu(+) chromosome later in evolution; and (3) expansion of CTG alleles occurred from (CTG)5 alleles on both Alu(+) and Alu(-) backgrounds.