High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes.

As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (> 500) simultaneously for a large number of known mutations (> 100) in a single assay. MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in > 500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion or deletion mutations. This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.

A unique nucleoprotein structure associated with the Drosophila melanogaster 18-28 S rDNA nontranscribed spacer.

We have detected unique nucleoprotein particles specific for the 18-28 S rDNA nontranscribed spacer of Drosophila melanogaster. The particles migrate between di- and trinucleosomes on nucleoprotein gels, and are between mono- and dinucleosomal in DNA length. These migration properties suggest that the nontranscribed spacer particles could have a protein component larger than a histone core. The variant nucleoprotein structures map primarily within the nontranscribed spacer 235 base pair internal subrepeat, which is AT-rich and possesses a 50 base pair sequence homologous to the RNA polymerase I binding site.

Induction of malignant transformation by various chemicals in Balb/3T3 clone A31-1-1 cells and biological characterization of some transformants.

Balb/c A31-1-1 cells were used for the study of transformation induction by chemicals with different mutagenic specificities. We show that survival of these cells and therefore the calculated transformation frequency per cells at risk is dependent upon the cell density at the time of treatment. It is suggested that equal cell densities should be used for measuring survival values and transformation induction. The quantitative results obtained are discussed in the light of the known mutagenic mechanisms of the chemicals tested. We also characterized morphologically transformed foci induced by different chemicals with respect to some biological properties. Anchorage independence was determined by testing growth in soft agar, loss of contact inhibition was quantitated by measuring maximum cell densities and malignancy was tested by tumor induction in nude mice. Although no very close correlation between these parameters and morphology was observed, the most malignant clones are also the ones with the highest values in the other tests. Our data make one or few genetical targets for transformation induction likely. We therefore speculate that the diverse phenotypes obtained might be due to differential activation of one or very few transforming genes in these cells.

Excretion of modified nucleosides during development of malignant lymphomas in mice after whole body irradiation.

During x-ray-induced development of malignant lymphomas in mice their urinary excretion of eight modified nucleosides was monitored and the values were compared to the results of the histological examination of the animals at time of their sacrifice. It was found that the pathologically augmented excretion of modified nucleosides begins as much as several weeks before the malignant lymphomas can be diagnosed clinically. Thus some mice had increased levels of modified nucleosides even 10 weeks before sacrifice, though at the time of sacrifice the histological investigation revealed only some small foci of reticulum cell neoplasm in their spleen. It is therefore stressed that the usefulness of the determination of urinary modified nucleosides as an early noninvasive screening test for cancer in man and as an in vivo carcinogenicity test should be evaluated.

A craniofacial growth maturity gradient for males and females between 4 and 16 years of age.

Differential growth of the craniofacial complex implies variation in ontogenetic patterns of development. This investigation quantifies the relative maturity--as defined by percent adult status--of nine cephalometric dimensions and stature. Analysis is based on 663 lateral cephalograms from a mixed longitudinal sample of 26 males and 25 females between 4 and 16 years of age. Graphic comparison of maturity status across the age range shows that variation is intergraded between the neural and somatic growth maturity patterns, as described by head height and stature, respectively. The maturity gradient moves from head height through anterior cranial base, posterior cranial base and maxillary length, upper facial height, corpus length, and ramus height to stature. After 9 years of age ramus height is less mature than stature. Anterior maxillary and mandibular heights diminish during transitional dentition and thereafter exhibit maturity patterns that compare to corpus length. Although females are consistently more mature than males, the gradient of variation between dimensions is sex independent.

Evaluation of a mutation test using S49 mouse lymphoma cells and monitoring simultaneously the induction of dexamethasone resistance, 6-thioguanine resistance and ouabain resistance.

A test for the detection of chemically induced mutants in S49 mouse lymphoma cells is described. These cells can be plated in parallel in several selective media; the induced frequencies of dexamethasone-resistant, 6-thioguanine-resistant and ouabain-resistant mutants were compared. The first two selection systems permit the detection of all kinds of mutation that result in alteration or partial or complete loss of the gene product concerned, whereas ouabain-resistant mutants can only be induced with strong point mutagens in these cells. Dexamethasone resistance is the marker induced at the highest frequency among these three. Data obtained from this selection system are therefore the most amenable to statistical analysis. Dexamethasone resistance is expressed within a short time after mutagenesis (3 days), and because S49 cells do not display metabolic co-operation, large numbers of cells can be screened. A metabolizing system in vitro with rat-liver homogenate may be included in tests of indirectly acting mutagens. These features make the S49 mutation test system using dexamethasone resistance as the main marker and other markers as internal controls an attractive tool in mutation testing in somatic cells in vitro.

Increasing urinary levels of modified nucleosides and bases during tumor development in mice.

Based on the fact that human cancer patients excrete increased amounts of various modified nucleosides and bases in their urine, we investigated whether the same phenomenon takes place in mice bearing experimentally induced tumors. We did indeed find that mice with MCA-induced skin tumors and mice exhibiting leukemia after X-ray irradiation excrete severalfold higher levels of modified nucleosides and bases than do untreated control mice. Comparison of the time course of altered urinary excretion of these RNA catabolites with the appearance of a recognizable tumor after MCA application revealed that the onset of the altered excretion rate of these compounds precedes tumor diagnosis. At present, the time-course studies in our mice exposed to a single X-ray dose to induce lymphoblastic leukemia seem to indicate a similar situation. Mice exhibiting preleukemic histological features already excrete increased amounts of various modified nucleosides and bases. Confirmation of our results by analysis of further irradiation-exposed mice in our present detailed time-course studies and of tumors experimentally induced in other organs of mice and other species will be taken as a basis for developing an in vivo test for carcinogenicity. Furthermore, the results could provide a foundation for the setting up of a noninvasive, early screening method for cancer in human beings.

Increased urinary levels of RNA catabolites in mice as early indicators for malignant growth.

It is known that human cancer patients exhibit an altered urinary excretion pattern of modified nucleosides and bases, that mice bearing skin tumors excrete increased amounts of various modified nucleosides and bases, and that the onset of altered excretion of modified RNA constituents precedes tumor diagnosis. We have now obtained similar results in mice for lymphoblastic leukemia induced by a single exposure to X-ray irradiation. Mice showing severe leukemic symptoms excrete severalfold amounts of modified nucleosides in their 24-hr urine, when compared with untreated controls. X-ray-treated mice, showing no visible symptoms except increased excretion rates of these RNA constituents, were sacrificed and histologically examined. (Pre)leukemic features in spleen and lymph nodes were observed. Our results provide further evidence in support of the use of RNA catabolites as a basis for the development of an early noninvasive screening method for cancer in human beings.

Craniofacial growth and size patterns during postnatal development.

Patterns of craniofacial growth and size relationships are described for a mixed longitudinal sample of 26 males and 25 females, followed serially from four years of age through adult status. The seven dimensions examined, derived from 663 lateral cephalograms, show differential patterns of negative allometric growth relative to statural increase. Relative growth is greatest for mandibular traits, followed by upper facial and neurocranial traits, respectively. Males exhibit greater relative growth than females. Craniofacial variation residual to allometry follows three independent patterns of association defined by principal component analysis. The components are age and sex independent, suggesting that after proportional changes related to absolute size or scale are controlled for, anterior facial, cranial height, and masticatory associations follow separate but proportionate patterns during growth.

Principal components of craniofacial growth for white Philadelphia males and females between 6 and 22 years of age.

Three principal components, explaining 83 percent of the common variation for 999 males and females between 6 and 22 years of age, describe ontogenetic patterns of relationship for seven facial dimensions, including sella-nasion, sella-basion, nasion-prosthion, infradentale-menton, articulare-gnathion, gonion-gnathion, and articulare-gonion. Accounting for 65 percent of the variation, a general component associated with both size and shape defines size-required changes in proprotion during growth. Independent patterns of regional variation associated with alveolar remodeling (second component) and condylar growth (third component) describe specific sources of facial modification. Mean multivariate component scores reveal that sexual dimorphism, which progressively favors males over females with age, results from accumulating differences in size and related proportional changes in shape. The timing of the condylar growth spurt, as evident from variation in ramus height, produces secondary dimorphism which diminishes following the adolescent phase in males. Significant age effects are indicated for alveolar remodeling and mandibular growth of the condyle.

Elevated urinary excretion of RNA catabolites as an early signal of tumor development in mice.

Determination of the urinary excretion rates of 12 modified nucleosides and bases in mice after tumor induction by application of a single dose of 3-methylcholanthrene revealed that mice bearing tumors in advanced stages excrete many-fold amounts of these nucleic acid catabolites compared with the control values. The excretion rate of several of these nucleic acid constituents like ac4C, m1A, PsU, and m2Gua increased before the tumor was diagnosable. Untreated control mice as well as mice having received the carcinogen, but not developing a tumor, did not show an alteration in the excretion values of any of the modified nucleosides and bases determined.

Genetic analysis of mutations causing borrelidin resistance by overproduction of threonyl-transfer ribonucleic acid synthetase.

Mutations leading to borrelidin resistance in Escherichia coli by overproduction of threonyl-transfer ribonucleic acid synthetase were anaylzed genetically. The regulatory mutations were closely linked to the treonyl-transfer ribonucleic acid synthetase structural gene (thrS), located clockwise to it. The mutation that causes the threefold-increased enzyme level was more distant from thrS than the mutation responsible for the ninefold overproduction. Both mutations were cis dominant in merodiploid strains, indicating that they affected promoter-operator-like control elements. Overproduction was restricted to threonyl-transfer ribonucleic acid synthetase and was not observed for the products of genes neighboring thrS (e.g., infC, pheS, pheT, and argS), providing evidence that thrS is transcribed singly and that gene amplificationis not a likely basis for increased thrS experession.

Non-coordinate regulation of enzymes involved in transfer RNA metabolism in Escherichia coli.

During different steady state growth conditions in Escherichia coli the level of the three tRNA-modifying enzymes, the tRNA(m5Urd)-, tRNA(m1Guo)- and tRNA(mam5s2Urd)methyltransferase and of five aminoacyl-tRNA synthetases, the leucyl-, valyl-, isoleucyl-, arginyl- and threonyl-tRNA-synthetase, has been determined. It is shown that those two classes of tRNA affecting enzymes are not coordinately regulated and that even within these two groups of enzymes the constituents are regulated independently of each other. Furthermore it is demonstrated that none of the aminoacyl-tRNA synthetases and only one of the three tRNA-methyltransferases, the tRNA(m5Urd)methyltransferase, is under control of the relA+-gene.

Escherichia coli mutants overproducing phenylalanyl- and threonyl-tRNA synthetase.

The structural genes for threonyl-tRNA synthetase (ThrRS) and phenylalanyl-tRNA synthetase (PheRS) are closely linked on the Escherichia coli chromosome. To study whether these enzymes share a common regulatory element, we have investigated their synthesis in mutants which were selected for overproduction of either ThrRS or PheRS. It was found that mutants isolated previously for overproduction of ThrRS as strains resistant to the antibiotic borrelidin (strains Bor Res 3 and Bor Res 15) did not show an elevated level of PheRS. PheRS-overproducing strains were then isolated as revertants of strains with structurally altered enzymes. Strain S1 is a temperature-resistant derivative of a temperature-sensitive PheRS mutant, and strain G118 is a prototrophic derivative of a PheRS mutant which shows phenylalanine auxotrophy as a consequence of an altered K(m) of this enzyme for the amino acid. In both kinds of revertants, S1 and G118, the concentration of PheRS and ThrRS was increased by factors of about 2.5 and 1.8, respectively, whereas the level of other aminoacyl-tRNA synthetases was not affected by the mutations. Genetic studies showed that the simultaneous overproduction of PheRS and ThrRS in revertants G118 and S1 is based upon gene amplification, since this property was easily lost after growing the cells in the absence of the selective stimulus, and since this loss could be prevented by the presence of the recA allele. By similar criteria, the four- and eightfold overproduction of ThrRS in strains Bor Res 3 and Bor Res 15, respectively, was very stable genetically, indicating that it is caused by a mutational event other than gene amplification. From these results, we conclude that the concomitant increase of PheRS and ThrRS in strains G118 and S1 is an expression of gene duplication and not of a joint regulation of these two aminoacyl-tRNA synthetases. This conclusion is further supported by the result that, in mutant G118 as well as in its parental strain G1, growth in minimal medium lacking phenylalanine led to an additional twofold increase of their PheRS concentration. This increase was restricted to the PheRS, since the level of other aminoacyl-tRNA synthetases, including the ThrRS, stayed unchanged.

Alteration of the intracellular concentration of aminoacyl-tRNA synthetases and isoaccepting tRNAs during amino-acid limited growth in Escherichia coli.

Growth of Escherichia coli AB 2271 under threonine or isoleucine deficiency leads to a depression of the threonyl-tRNA synthetase and isoleucyl-tRNA synthetase respectively. During this amino-acid-limited growth the concentrations of isoaccepting fractions of the cognate tRNA species were changed, as demonstrated by their altered reversed-phase-5 chromatograms. But, in addition, the profiles of the isoacceptors of all other tRNA species investigated, i.e. of tRNAsLeu, tRNAsSer and tRNAsArg were also altered. This means that, if there is a correlation between regulation of the level of an aminoacyl-tRNA synthetase and its cognate isoaccepting tRNAs, it is superimposed by the effect of amino acid limitation upon the concentration of all isoaccepting tRNAs. So far drastic changes in profiles of isoaccepting tRNAs have only been observed under unbalanced growth in relaxed cells or during treatment with antibiotics. Here we demonstrate that similar heavy alterations in patterns of isoaccepting tRNAs occur in a proven stringent E. coli strain growing exponentially under amino acid limitation. Thus the observed changes in the profiles of isoaccepting tRNAs during amino acid limitation signal a meaningful biological function of those newly or increasingly occurring isoaccepting tRNAs. During the growth under amino acid limitation the total acceptor activity of eight investigated tRNA species, however, stayed unchanged, except that under threonine-limited growth the total amount of tRNAIle was reduced to about half and that of tRNAGlu increased; both tRNA species of these isoacceptors are known [30,31] as spacers between ribosomal RNAs.

Genetically determined differences in concentrations of isoaccepting tRNAs in Escherichia coli.

Two examples of genetically determined altered concentrations of isoaccepting tRNAs are presented. The concentrations of isoaccepting tRNAsThr are selectively changed by a mutation causing a fourfold overproduction of the cognate aminoacyl-tRNA-synthetase, the threonyl-tRNA synthetase, whereas the distribution of isoaccepting tRNAs of four control tRNA-species in these E. coli mutants was not affected by that mutation. Secondly evidence is presented for a correlation between mutations in structural genes of aminoacid biosynthetic enzymes and alterations in concentrations of cognate isoaccepting tRNAs in two different E. coli strains, auxotrophic for threonine, isoleucine/valine and leucine, and arginine respectively.

Threonyl-transfer ribonucleic acid synthetase from Escherichia coli: subunit structure and genetic analysis of the structural gene by means of a mutated enzyme and of a specialized transducing lambda bacteriophage.

Threonyl-transfer ribonucleic acid synthetase (ThrRS) has been purified from a strain of Escherichia coli that shows a ninefold overproduction of this enzyme. Determination of the molecular weight of the purified, native enzyme by gel chromatography and by polyacrylamide gel electrophoresis at different gel concentrations yielded apparent molecular weight values of 150,000 and 161,000, respectively. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate yields a single protein band of 76,000-dalton size. From these results an alpha(2) subunit structure can be inferred. A mutant with a structurally altered ThrRS, which had been obtained by selection for resistance against the antibiotic borrelidin, was used to map the position of the ThrRS structural gene (thrS) by P1 transductions. It was found that thrS is located in the immediate neighborhood of pheS and pheT, which are the structural genes for the alpha and beta subunits of phenylalanyl-transfer ribonucleic acid (tRNA) synthetase, the gene order being aroD-pheT-pheS-thrS. A lambda phage that was previously shown to specifically transduce pheS, pheT, and also the structural gene for the translation initiation factor IF3 can complement the defect of the altered ThrRS of the borrelidin-resistant strain. This phage also stimulates the synthesis of the 76,000, molecular-weight polypeptide of ThrRS in ultraviolet light-irradiated. E. coli cells. These results indicate that the genes for ThrRS, alpha and beta subunits of phenylalanyl-tRNA synthetase, and initiation factor IF3 are immediately adjacent on the E. coli chromosome.

Intra-group variations in the dental eruption sequence of Macaca fuscata fuscata.

The purpose of this investigation was to establish general trends of intra-group variations in the dental eruption sequence of a semi-wild troop of Maca fuscata fuscata. Dental impressions were taken on 128 animals of varying ages. The analysis of the dental casts revealed sexual dimorphism in sequence and timing of eruption.