Recessive Nonsense Suppressors in SACCHAROMYCES CEREVISIAE : Action Spectra, Complementation Groups and Map Positions

Three genes SUP111, SUP112 and SUP113 of Saccharomyces cerevisiae have been identified that can mutate to give recessive omnipotent nonsense suppressors. Alleles of these loci can also act as allosuppressors; that is, different phenotypes, due apparently to different efficiencies of suppression, can result from different alleles at a given locus. The SUP111, SUP112 and SUP113 loci map to the right arms of chromosomes VIII, VII and XIII, respectively.     

Induction of gametocytogenesis in Plasmodium falciparum by the culture supernatant of hybridoma cells producing anti-P. falciparum antibody

There have been many unsuccessful attempts to induce gametocytogenesis in vitro. In the present experiment, however, we found that RPMI-CS medium and RPMI-FS medium prepared by dissolving powdered RPMI 1640 medium in the culture supernatants of hybridoma cells, hybrid line D21 and 219.5, respectively, that produce anti-P. falciparum antibody induced gametocytogenesis. Gametocytogenesis was consistently observed from 3 days after addition of these media. The culture supernatant of anti-P. falciparum antibody producing hybridoma cells did not induce gametocytogenesis in the absence of RPMI 1640 medium. RPMI-MS medium, prepared by dissolving powdered RPMI 1640 medium in the culture supernatant of myeloma cells, SP2/O-Ag 14, which was used as a control, induced a few gametocytes.     

Organization and evolution of variable region genes of the human immunoglobulin heavy chain

We have isolated 23 different cosmid clones of the heavy-chain variable region genes (VH) of human immunoglobulin. These clones encompass about 1000 X 10(3) base-pairs of DNA containing 61 VH genes. Characterization of the 23 clones by Southern blot hybridization showed that VH genes belonging to different families were physically linked in many regions. Cluster 71, which was analyzed in detail, comprised seven VH segments arranged in the same orientation with different intervals. This clone contained internal homology regions, each carrying two VH segments of different families. Comparison of the nucleotide sequences of VH segments within each family showed that profiles of accumulation of mutations in framework (FR) and complementarity-determining (CDR) regions were different. CDR had more mutations at amino-acid-substituting positions than at silent positions, whereas FR had the reverse distribution of mutations. Five out of seven VH segments of this cluster were pseudogenes containing various mutations. VH pseudogenes were classified into two distinct groups; one with a few replacement mutations (conserved pseudogenes), and the other with rather extensive mutations (diverged pseudogenes). The possibility that conserved pseudogenes serve as a reservoir of VH segments is discussed.     

Immunological characterization of sn-1,2-diacylglycerol and sn-2-monoacylglycerol kinase from pig brain

Rabbit antisera were raised against diacylglycerol kinase purified from pig brain cytosol. Upon immunoblot analysis, the antibody was specifically reactive with the kinase (Mr = 79,000-80,000). Pig brain cytosol, microsomal, and synaptosomal fractions all contained the immunoreactive Mr = 80,000 polypeptide, thus showing that the same enzyme is present in the soluble as well as membrane fractions of the brain. The antibody could precipitate only 60% of the kinase activity present in the crude cytosol. Further, the antibody exhibited very little or no cross-reactivity toward liver cytosolic enzymes obtained from different animals including pigs. Immunostaining of brain tissues demonstrated that neurons, in particular, their nuclei, were positively stained, whereas glial cells were not stained. It is likely that there exists a tissue-and/or cell-dependent immunological multiplicity of diacylglycerol kinase. The enzyme activities phosphorylating sn-1 and sn-2 monoacylglycerols were co-precipitated by the antibody, indicating their identity with diacylglycerol kinase. The enzyme activity toward sn-1 monoolein was much lower than that obtained with sn-2 monoolein. Enzymic as well as chemical analyses of acyl isomers of the reaction products showed that even tested with pure (greater than 95%) sn-1 monoolein, about 70% of the formed lysophosphatidate was of the sn-2 acyl type. The results show that diacylglycerol kinase phosphorylates almost exclusively the sn-2 acyl type of monoacyl-glycerol.     

Fever in rats during normal and dehydrated conditions

The effect of endogenous pyrogen (EP, from rabbit) and endotoxin (Salmonella typhosa) on rectal temperature (Tre) was investigated in normal and dehydrated rats of both sexes. Intraperitoneal injection of either EP or endotoxin did not affect body temperature. In addition, no changes in Tre were observed when endotoxin was injected intravenously in normally hydrated male rats, but significant falls in Tre occurred in normal female rats. However, intravenous injection of EP produced fever in both sexes, but females generally showed smaller responses. A second intravenous injection of endotoxin, given 3 days after the first injection, always produced fever in normally hydrated rats. The pattern of this febrile response was monophasic. In contrast to the response in normal rats, intravenous endotoxin produced significant fevers with a biphasic pattern in dehydrated rats of either sex, but the febrile responses of male rats were greater than those of female rats. On the other hand, there were no significant differences between febrile responses to intravenous EP exhibited by normal and dehydrated animals. These results show that rats of both sexes possess physiological mechanisms capable of producing a fever following intravenous injections of EP.     

Protein phosphorylation of beta-glucuronidase in human lung cancer--identification of serine- and threonine-phosphates

Slices of human lung cancer tissue were incubated with [32P]-orthophosphoric acid, and the radiolabeled beta-glucuronidase was isolated by a procedure including immunoaffinity chromatography on anti-human liver beta-glucuronidase IgG Sepharose. Following removal of endo-beta-N-acetyl-glucosaminidase H-releasable carbohydrate portions of the enzyme, the protein moiety was acid-hydrolyzed. Two-dimensional separation of the hydrolysate identified phosphoserine and phosphothreonine. This is the first demonstration of protein phosphorylation in lysosomal beta-glucuronidase.     

Fucosylation of serum alpha-fetoprotein in patients with primary hepatocellular carcinoma

alpha-Fetoprotein specimens were prepared from the sera of four patients with hepatocellular carcinoma. The lentil lectin-reactive and lectin-nonreactive variants of this glycoprotein were also prepared from the serum of one of the four patients by affinity chromatography with immobilized lectin. The correlation between the carbohydrate structure of these compounds and their reactivity in crossed immuno-affinoelectrophoresis with lentil lectin was studied by chemical analysis and affinity chromatography of the glycopeptides with lectin columns. It was found that the lentil lectin-reactive variant contained a carbohydrate chain of the fucosylated biantennary complex type. These data together with previous findings indicate that most of the patients with hepatocellular carcinoma have an elevated serum concentration of fucosylated alpha-fetoprotein.     

Defective endocytosis of low-density lipoprotein in monensin-resistant mutants of the mouse Balb/3T3 cell line

Two monensin-resistant clones show similar low-density lipoprotein binding activity but less internalization or degradation of low-density lipoprotein than the parental Balb/3T3 or other resistant clone. Sterol synthesis from radioactive acetate in the resistant mutant, MO-5, is inhibited by more than 70% of control in the presence of tenfold higher amounts of low-density lipoprotein than the dose that inhibits the parental Balb/3T3 to similar level. 3-Hydroxy-3-methylglutaryl coenzyme A reductase activity of Balb/3T3 and MO-5 is inhibited by 48% and 27% of control, respectively, in the presence of 10 micrograms/ml of low-density lipoprotein. Colloidal silica gradient centrifugation shows that transport of low-density lipoprotein from the surface membrane to the lysosome is much slower in MO-5 cells than in Balb/3T3 cells. Down regulation of low-density lipoprotein receptors on the cell surface in Balb/3T3 is observed by exposing the cells to 5-15 micrograms/ml low-density lipoprotein, whereas only slight if any down regulation is observed when MO-5 cells are treated with low-density lipoprotein. The altered endocytosis of low-density lipoprotein behaves as a dominant trait in hybrids of MO-5 and THO2-2, a derivative of Balb/3T3 resistant to both ouabain and 6-thioguanine.     

Nucleotide sequence of the Syrian hamster intracisternal A-particle gene: close evolutionary relationship of type A particle gene to types B and D oncovirus genes.

We determined the complete nucleotide sequence of the intracisternal A-particle gene, IAP-H18, cloned from the normal Syrian hamster liver DNA. IAP-H18 was 7,951 base pairs in length with two identical long terminal repeats of 376 base pairs at both ends. On the coding strand, imperfect open reading frames corresponding to gag and pol of the retrovirus genome were observed, whereas many stop codons were present in the region corresponding to env. The putative H18 gag gene (809 amino acids) had a sequence homologous to the N-terminal half of the mouse mammary tumor virus gag gene and locally to the Rous sarcoma virus gag gene. The putative H18 pol gene (900 residues) was homologous to the Rous sarcoma virus pol gene almost throughout the entire region. Two conserved regions among the retrovirus pol genes have been reported. One presumably corresponds to the DNA polymerase and the RNase H domain, and the other corresponds to the DNA endonuclease domain of the multifunctional protein pol. By the comparison of the deduced amino acid sequences of the putative endonuclease domain of six representative oncovirus genomes, a phylogenetic tree of the oncovirus genomes was constructed, and the intracisternal A-particle (type A) genome was found to be more closely related to the mouse mammary tumor virus (type B) and squirrel monkey retrovirus (type D) genomes.     

Fine-granular cationic iron colloid. Its preparation, physicochemical characteristics and histochemical use for the detection of ionized anionic groups

In order to obtain distinct and reliable information concerning the localization of ionized anionic groups in tissues, fine-granular cationic ferric hydroxide colloid solution (Fe-Cac-f) was newly devised. This can be obtained by boiling a mixture of ferric chloride and ammonium cacodylate solutions. The colloid particles of Fe-Cac-f are about 1.0 nm in size, i.e., one-fifth of the size of ferric cacodylate colloid (Fe-Cac; Seno et al. 1983a). As with Fe-Cac, Fe-Cac-f particles in the pH range of 1.6-7.6 carry a positive electric charge, but the latter show a better permeation of tissues. Using the Prussian blue reaction, Fe-Cac-f gives a distinct deep-blue color and can be used for the detection of anionic groups of acid mucopolysaccharides and proteins by light microscopy. It is also useful for detecting the exact sites of ionized anionic groups in deep tissue areas using electron microscopy.