Highly frequent single amino acid substitution in mammalian elongation factor 2 (EF-2) results in expression of resistance to EF-2-ADP-ribosylating toxins

Toxin-resistant polypeptide chain elongation factor 2 cDNA has been cloned from a mutant hamster cell line with only non-ADP-ribosylatable elongation factor 2. The mutation conferring resistance to diphtheria toxin and Pseudomonas aeruginosa exotoxin A is a G-to-A transition in the first nucleotide of codon 717. Codon 715 encodes a histidine residue that is modified post-translationally to diphthamide, which is the target amino acid for ADP-ribosylation by both toxins. Transfection of mouse L cells with a recombinant elongation factor 2 cDNA differing from the wild-type only by this G-to-A transition confers resistance to P. aeruginosa exotoxin A. The degrees of toxin-resistant protein synthesis of stable transfectants are dependent on the ratio of non-ADP-ribosylated elongation factor 2 to wild-type elongation factor 2, not the amount of non-ADP-ribosylated elongation factor 2. The mutation creates a new Mbo II restriction site in the elongation factor 2 gene. Several independently isolated diphtheria toxin-resistant Chinese hamster ovary cell lines show the same alteration in the Mbo II restriction pattern.     

The mode of entry of herpes simplex virus type 1 into Vero cells

The mode of entry of herpes simplex virus type 1 (HSV-1) into Vero cells was investigated quantitatively with biological techniques. The entry of virus occurred rapidly when the virus-adsorbed cells were incubated at 37 C. The kinetics of virus entry was found to be similar to that of the process of uncoating, indicating that the uncoating of HSV-1 occurs simultaneously with the entry of virus into the cell. Experiments with ammonium chloride revealed that acidity in endosomes is not necessary for the entry or uncoating of HSV-1, in contrast with the cases of enveloped RNA viruses. In addition, endocytosis of the virus seems to be one of the processes of entry for HSV-1. However, the kinetics of endocytosis showed that the cell-bound virus is endocytosed gradually and suggested that the endocytosis of HSV-1 does not lead the virus to an uncoating process. These results are most consistent with a mechanism of entry for HSV-1 involving fusion of the viral envelope with the plasma membrane of the host cell.     

Evolutionary relationships between laboratory mice and subspecies of Mus musculus based on the genetic study of pancreatic proteinase loci,Prt-1, Prt-2, Prt-3, and Prt-6

Various patterns of mouse pancreatic proteinase activity bands were observed on agarose gel electrophoresis. Prt-1 ^a and Prt-1 ^b genes control the positive (PRT-1A) and negative (PRT-1B) expression of tryptic band V, respectively; Prt-2 ^a and Prt-2 ^b correspond to chymotryptic bands II (PRT-2A) and III (PRT-2B); Prt-3 ^a and Prt-3 ^b control the low (PRT-3A) and high (PRT-3B) tryptic activities of band IV; the Prt-1 and Prt-3 loci are closely linked on the same chromosome; Prt-6 ^a and Prt-6 ^b correspond to tryptic bands I (PRT-6A) and I (PRT-6B). Twenty-four laboratory strains from the United States showed the phenotype PRT-1A, PRT-3A, and PRT-2A. Of laboratory strains established in Europe, 6 showed PRT-1A, PRT-3A, and PRT-2A, and 10 had PRT-1B, PRT-3A, and PRT-2A bands. Most wild mice around the world and their descendants showed the phenotype PRT-1B, PRT-3B, and PRT-2A. Only the phenotype of M. m. brevirostris was PRT-1A, PRT-3A, and PRT-2A, which was the same as most laboratory inbred strains. PRT-2B was observed mainly in Japanese (M. m. molossinus) and Korean (M. m. yamashinai) wild mice. PRT-6B was detected only in Mus spicilegus and Mus caroli, but all other mice including wild populations and laboratory strains showed PRT-6A. New biochemical phenotypes such as PRT-2C and PRT-3C were also found in this study.     

Regional assignment of the gene for diphtheria toxin sensitivity using subchromosomal fragments in microcell hybrids

Human x mouse microcell hybrids resistant to G418 were constructed between mouse hepatoma cells and human x mouse whole cell hybrids containing only intact human chromosome 5 and 22 with an integrated neo ^r-gene. Among these, microcell hybrid BG15 produced four subclones, BG15-4, BG15-6, BG15-7 and BG15-9, which contained variously sized complements of human chromosome 5. BG15-6 contained an intact human chromosome 5, BG15-7 a deleted human chromosome 5 (5pter-q22) and BG15-4 and BG15-9 a translocation between parts of human chromosome 5 (pter-qter? and pter-q23, respectively) and a mouse chromosome. Southern DNA blot analysis showed that the human dihydrofolate reductase (DHFR) gene was present in all four subclones, whereas the human homolog of the v-fms gene was present in BG15-4 and 15-6, but absent from BG15-7 and 15-9. BG15-4, 15-6 and 15-9 were sensitive to diphtheria toxin, and only BG15-7 was resistant to the toxin. We used these microcell hybrids to restrict further the regional location of the gene for diphtheria toxin sensitivity to the q23 region of human chromosome 5.     

Regional assignment of five genes on human chromosome 19

A human-mouse hybrid segregant HM76Dd40-6 with new characteristics was derived from the hybrid cell line HM76Dd containing human chromosome 19 as the only human chromosome. Three virus sensitivities located on human chromosome 19 (PVS, E11S and RDRC) were lost in HM76Dd40-6, while six other genes (C3, LDLR, EF2, GPI, PEPD and MANB) were retained. Cytogenetic analysis and in situ hybridization using human or mouse repeated sequences as probes showed that the region q13.1-qter of human chromosome 19 had been replaced by a fragment of mouse chromosome. Our results permit further regional assignment for the following five genes on human chromosome 19: GPI in the region cen-q12, MANB in p13.2-q12, E11S and RDRC in q13.1-qter, and EF2 in pter-q12.     

The Relationship between Dipeptidase Activity Variation and Larval Viability in Drosophila melanogaster

The enzyme dipeptidase-A (DIP-A) in Drosophila melanogaster is coded by a second chromosome locus that is polymorphic for three allozymes in natural populations. DIP-A appears to be the only enzyme in D. melanogaster capable of hydrolyzing the dipeptide glycyl-L-isoleucine, since flies homozygous for null alleles at this locus have no detectable glycyl-L-isoleucine-ase activity. DIP-A activity occurs in many tissues and throughout development, but is particularly high in the larval midgut, suggesting an important role in protein digestion. These observations suggested an experimental design for investigating the adaptive significance of genetic variation in DIP-A activity. Fitness components of DIP-A variants could be estimated and compared under two environmental conditions (defined diets under axenic conditions). In the restrictive environment, the essential amino acid L-isoleucine is provided only in the form of glycyl-L-isoleucine, whereas in the permissive environment, L-isoleucine is provided in free form. We predicted that DIP-A activity would be essential in the restrictive, but not in the permissive environment. The results reported here clearly contradict this prediction. Two stocks homozygous for DIP-A null alleles from different geographic locations are each viable on the restrictive diet. Furthermore, relative viability experiments in which null allele larvae compete with larvae having DIP-A activity provide no evidence for even a partial reduction in egg to adult survival on the restrictive diet. Apparently, the null allele larvae have some alternative mechanism for obtaining L-isoleucine from the dipeptide, even though no glycyl-L-isoleucine-ase activity can be detected in vitro. These results, along with the viability of null alleles for many other enzymes, support the idea that eukaryotes have an intricate network of alternative biochemical pathways through which the same necessary function may be achieved. Such "buffering capacity" makes it very difficult to analyze the effects of enzyme variants on fitness components.     

Effect of roasting on ochratoxin A level in green coffee beans inoculated with Aspergillus ochraceus

The heat stability of ochratoxin A in green coffee beans inoculated with Aspergillus ochraceus was studied. Heat treatment (roasting) at 200 °C for 10 or 20 min reduced the levels of ochratoxin A by only 0–12% in the dried whole beans. Almost all of the ochratoxin A was infused into the coffee decoction when the roasted samples were ground and extracted with boiling water. Therefore, the reduction of ochratoxin A concentration of contaminated coffee beans by roasting under these conditions is ineffective.     

Polymorphism of T-cell receptor genes among laboratory and wild mice: Diverse origins of laboratory mice

Southern blots of genomic DNA from 23 strains of laboratory mice and 19 individual wild mice were examined for restriction fragment length polymorphisms in their loci encoding the T-cell receptors (Tcr): the constant regions of the α, β, and γ chains (C _α,C _β, andC _γ) and a variable region family of the β chain (V _β8). Only a few polymorphisms were observed for each locus in the laboratory mice after using three restriction enzymes,Bam HI,Eco RI, andHind III. All the laboratory mice examined fall into one of two types for theC _α,C _β andV _β8 loci and one of three types for theC _γ. These types are found in some of the wild mice studied, indicating that they were already present in the founder mice of laboratory mouse strains. In contrast, theTcr genes are highly polymorphic among wild mice. Analysis of the polymorphisms in these loci suggests that laboratory mice have inherited their genes not only fromMus musculus domesticus, but also from other subspecies, and much more than previously believed from Asian subspecies.     

Conformational change of bovine serum albumin by heat treatment

The thermal denaturation of bovine serum albumin (BSA) was studied at pH 2.8 and 7.0 in the range of 2–65°C. The relative proportions of -helix, -structure, and disordered structure in the protein conformation were determined as a function of temperature, by the curve-fitting method of circular dichroism spectra. With the rise of temperature at pH 7.0, the proportion of -helix decreased above 30°C and those of -structure and disordered structure increased in the same temperature range. The structural change was reversible in the temperature range below 45°C. However, the structural change was partially reversible upon cooling to room temperature subsequent to heating at 65°C. On the other hand, the structural change of BSA at pH 2.3 was completely reversible in the temperature range of 2–65°C, probably because the interactions between domains and between subdomains might disappear due to the acid expansion. The secondary structure of disulfide bridges-cleaved BSA remained unchanged during the heat treatment up to 65°C at pH 2.8 and 7.0.