Characterization of Mutants of Escherichia coli Temperature-Sensitive for Ribonucleic Acid Regulation: an Unusual Phenotype Associated with a Phenylalanyl Transfer Ribonucleic Acid Synthetase Mutant

A mutant strain AA-522, temperature-sensitive for protein synthesis, was isolated from a stringent strain (CP-78) of Escherichia coli K-12. The mutant strain has a relaxed phenotype at the nonpermissive growth temperature. Protein synthesis stops completely at 42 C, whereas the rate of ribonucleic acid (RNA) synthesis is maintained at 20% of the 30 C rate. Sucrose-gradient centrifugation analysis of RNA-containing particles formed at 42 C indicated the presence of “relaxed particles.” These particles possess 16S and 23S RNA and are precursors to normal 50S and 30S ribosomal subunits. A search for the temperature-sensitive protein responsible for the halt in protein synthesis implicated phenylalanyl transfer RNA (tRNA) synthetase. Essentially no enzyme activity is detected in vitro at 30 or 40 C. Analysis of phenylalanyl tRNA synthetase activity in revertants of strain AA-522 indicated the presence of intragenic suppressor mutations. Revertants of strain AA-522 analyzed for the relaxed response at 42 C were all stringent; strain AA-522 was stringent at 30 C. These data indicate that a single mutation in phenylalanyl tRNA synthetase is responsible for both a block in protein synthesis and the relaxed phenotype at 42 C.     

Reactions of polycyclic hydrocarbon epoxides with RNA and polyribonucleotides

RNA, poly(G) and poly(A) were reacted with benz[a]anthracene 5,6-oxide or with 7,12-dimethylbenz[a]anthracene 5,6-oxide and hydrolysates of the alkylated polymers examined using a combination of Sephadex LH20 column chromatography and thin-layer chromatography on silica gel. The results show that two RNA products are formed in reactions with benz[a]anthracene 5,6-oxide, one resulting from reaction with guanine and the other from reaction with adenine. With 7,12-dimethylbenz[a]anthracene 5,6-oxide, six RNA products appeared to be formed, two resulting from reactions with guanine and three from alkylation of adenine; the other product has not been identified.     

Detection and Identification of Translocations by Increased Specific Nondisjunction in ASPERGILLUS NIDULANS

A meiotic technique for visual detection of translocations has been applied to ten mitotically identified interchanges, and three new translocations were discovered using this method. Testcrosses between "standard" strains and potential translocation strains-e.g. strains with newly induced mutants or descendants from translocation crosses-are inspected for the frequency of abnormal-looking colonies. In all heterozygous translocation crosses "abnormals" are increased at least tenfold compared to the average control level of 0.15%. Most of these are disomics, and can be recognized by their characteristic phenotypes. Each translocation produces a few specific types, since nondisjunction is increased mainly in the linkage groups involved in the translocation (50-100-fold over control values). Therefore, translocations were not only detected but often tentatively assigned to linkage groups from the analysis of the disomic progeny in crosses. In addition, this technique allows reciprocal and nonreciprocal translocations to be distinguished, since only the latter produce one-third phenotypically abnormal duplication progeny. While results are clearcut in most cases, occasionally problems are encountered, e.g. when morphological mutants segregate in crosses, or when other genetic factors which increase or reduce the frequency of nondisjunction are present in certain strains.     

Structural studies of an Inv (1, −2) kappa light chain

A Bence Jones protein with phenotype Inv (1, –2) was isolated from the urine of a patient with multiple myeloma. Inv typing of the patient's relatives established the presence of anInv ¹ allele in the kindred, and that the patient's genotype wasInv ¹/Inv ³. Hence, the absence of Inv (2) in the Bence Jones protein was shown to be genetic and not an artifact caused by the disease. The tryptic peptide-containing residues 191 through 194 were isolated and shown to be composed of Leu, Tyr, Ala, Cys, with Leu at the amino end. Hence, the residue at 191 is the same as that present in Inv (1, 2) Bence Jones proteins. More detailed study of the tryptic peptides established that residue 153 is Val rather than Ala as in all other K chains thus far studied. The primary sequence: Ala¹⁵³, Leu¹⁹¹ determines Inv (1, 2); Ala¹⁵³, Val¹⁹¹ determines Inv (3); and Val¹⁵³, Leu¹⁹¹ determines Inv (1). The Val¹⁵³, Val¹⁹¹ sequence has not been observed. It may correspond to Inv (–). These data are strikingly similar to the data for the Kern and Oz isotypes (changes at 154 and 191, respectively) in the chain. As in the case of theK chain, only three of the four possible combinations have been observed. The implications of this parallelism and of crystallographic findings on chains, reported by others, are discussed.     

Analysis of selection in populations observed over a sequence of consecutive generations

Summary A statistical model is presented for dealing with genotypic frequency data obtained from a single population observed over a run of consecutive generations. This model takes into account possible correlations that exist between generations by conditioning the marginal probability distribution of any one generation on the previously observed generation. Maximum likelihood estimates of the fitness parameters are derived and a hypothesis testing framework developed. The model is very general, and in this paper is applied to random-mating, selfing, parthenogenetic and mixed random-mating and selfing populations with respect to a single locus, g-allele model with constant genotypic fitness differences with all selection occurring either before or after sampling. The assumptions behind this model are contrasted with those of alternative techniques such as minimum chi-square or unconditional maximum likelihood estimation when the marginal likelihoods for any one generation are conditioned only on the initial conditions and not the previous generation. The conditional model is most appropriate when the sample size per generation is large either in an absolute sense or in relation to the total population size. Minimum chi-square and the unconditional likelihood are most appropriate when the population size is effectively infinite and the samples are small. Both models are appropriate when the samples are large and the population size is effectively infinite. Under these last conditions, the conditional model may be preferred because it has greater robustness with respect to small deviations from the underlying assumptions and has a greater simplicity of form. Furthermore, if any genetic drift occurs in the experiment, the minimum chi-square and unconditional likelihood approaches can create spurious evidence for selection while the conditional approach will not. Worked examples are presented.This study was supported in part by the U. S. Atomic Energy Commission, Contract AT (11-1) -1552 to the Department of Human Genetics (CFS), University of Michigan, and by National Science Foundation Grant BMS 74-17453 awarded to the author.     

Isolation of salt-soluble elastin from ligamentum nuchae of copper-deficient calf

This paper describes the isolation and amino acid analysis of un-cross-linked elastin obtained by neutral salt extraction from the ligamentum nuchae of a calf fed from birth to 9 months on a diet low in copper.     

Preliminary characterization of Thy-1.1 and Ag-B antigens from rat tissues solubilized in detergents

1. A radioactive binding assay for Thy-1.1 alloantigen which functions in the presence of detergents was established by using glutaraldehyde-fixed thymocytes as target cells. Thy-1.1 activity in detergent extracts was then assayed by measuring inhibition of the binding assay. 2. Solubilization of Thy-1.1 from whole thymocytes, and their membranes by a large number of non-ionic detergents and deoxycholate was studied. In the same extracts Ag-B(4) histocompatibility antigenic activities were measured. With the exception of Nonidet P-40, the detergents did not affect the antigenicity of Thy-1.1, but only Lubrol-PX and deoxycholate gave effective solubilization as measured by activity remaining in the supernatant after centrifugation at 200000g for 40min. With Ag-B(4) antigen, Triton X-100, Triton X-67 and Nonidet P-40 gave effective solubilization as well as Lubrol-PX and deoxycholate. Solubilization of Thy-1.1 activity from leukaemia cells and a brain homogenate was also studied, but none of the non-ionic detergents gave satisfactory results with these tissues. 3. Extracts from thymocyte membranes were further examined by gel filtration and sucrose gradient centrifugation. The Thy-1.1 activity behaved as a single component in deoxycholate with a density similar to that of a globular protein, but in Lubrol-PX the antigen was contained in a low-density complex. In Lubrol-PX extracts Ag-B(4) was also found in aggregates not observed in deoxycholate. 4. The s(20,w) values for Thy-1.1 and Ag-B(4) antigens in deoxycholate were 2.4 and 4.4, and v values were 0.70 and 0.75 respectively. The Stokes radius observed for Thy-1.1 was 3.1nm and for Ag-B(4) 5.3nm. By using these values the molecular weights for the antigen-detergent complexes were calculated to be 28000 for Thy-1.1 and 100000 for Ag-B(4).