Temperature-Sensitive Lethal Mutations on Yeast Chromosome I Appear to Define Only a Small Number of Genes

A method was developed for isolating large numbers of mutations on chromosome I of the yeast Saccharomyces cerevisiae. A strain monosomic for chromosome I (i.e., haploid for chromosome I and diploid for all other chromosomes) was mutagenized with either ethyl methanesulfonate or N-methyl-N'-nitro-N -nitrosoguanidine and screened for temperature-sensitive (Ts^- ) mutants capable of growth on rich, glucose-containing medium at 25° but not at 37°. Recessive mutations induced on chromosome I are expressed, whereas those on the diploid chromosomes are usually not expressed because of the presence of wild-type alleles on the homologous chromosomes. Dominant ts mutations on all chromosomes should also be expressed, but these appeared rarely. — Of the 41 ts mutations analyzed, 32 mapped on chromosome I. These 32 mutations fell into only three complementation groups, which proved to be the previously described genes CDC15, CDC24 and PYK1 (or CDC19). We recovered 16 or 17 independent mutations in CDC15, 12 independent mutations in CDC24 and three independent mutations in PYK1. A fourth gene on chromosome I, MAK16, is known to be capable of giving rise to a ts-lethal allele, but we recovered no mutations in this gene. The remaining nine mutations isolated using the monosomic strain appeared not to map on chromosome I and were apparently expressed in the original mutants because they had become homozygous or hemizygous by mitotic recombination or chromosome loss. — The available information about the size of chromosome I suggests that it should contain approximately 60–100 genes. However, our isolation in the monosomic strain of multiple, independent alleles of just three genes suggests that only a small proportion of the genes on chromosome I is easily mutable to give a Ts^--lethal phenotype. — During these studies, we located CDC24 on chromosome I and determined that it is centromere distal to PYK1 on the left arm of the chromosome.     

Electrogenic Cl− absorption byAmphiuma small intestine: Dependence on serosal Na+ from tracer and Cl− microelectrode studies

Summary The Na⁺ requirement for active, electrogenic Cl^– absorption byAmphiuma small intestine was studied by tracer techniques and double-barreled Cl^–-sensitive microelectrodes. Addition of Cl^– to a Cl^–-free medium bathingin vitro intestinal segments produced a saturable (K _m =5.4mm) increase in shortcircuit current (I _sc) which was inhibitable by 1mm SITS. The selectivity sequence for the anion-evoked current was Cl^–=Br^–>SCN^–>NO ₃ ^– >F^–=I^–. Current evoked by Cl^– reached a maximum with increasing medium Na concentration (K _m =12.4mm). Addition of Na⁺, as Na gluconate (10mm), to mucosal and serosal Na⁺-free media stimulated the Cl^– current and simultaneously increased the absorptive Cl^– flux (J _ms ^Cl ) and net flux (J _net ^Cl ) without changing the secretory Cl^– flux (J _sm ^Cl ). Addition of Na⁺ only to the serosal fluid stimulatedJ _ms ^Cl much more than Na⁺ addition only to the mucosal fluid in paired tissues. Serosal DIDS (1mm) blocked the stimulation. Serosal 10mm Tris gluconate or choline gluconate failed to stimulateJ _ms ^Cl . Intracellular Cl^– activity (a _Cl ⁱ ) in villus epithelial cells was above electrochemical equilibrium indicating active Cl^– uptake. Ouabain (1mm) eliminated Cl^– accumulation and reduced the mucosal membrane potential _m over 2 to 3 hr. In contrast, SITS had no effect on Cl^– accumulation and hyperpolarized the mucosal membrane. Replacement of serosal Na⁺ with choline eliminated Cl^– accumulation while replacement of mucosal Na⁺ had no effect. In conclusion by two independent methods active electrogenic Cl^– absorption depends on serosal rather than mucosal Na⁺. It is concluded that Cl^– enters the cell via a primary (rheogenic) transport mechanism. At the serosal membrane the Na⁺ gradient most likely energizes H⁺ export and regulates mucosal Cl^– accumulation perhaps by influencing cell pH or HCO ₃ ^– concentration.     

α-Ketoglutarate and Malate Uptake and Metabolism by Synaptosomes: Further Evidence for an Astrocyte-to-Neuron Metabolic Shuttle

This study was undertaken to provide further evidence relevant to the hypothesis that astrocytes supply one or more citric acid cycle intermediates to synaptic terminals, thereby serving an anaplerotic function necessitated by the synthesis and release of amino acid neurotransmitters. In our experiments, two populations of synaptosomes obtained from the brain of rats were separated from myelin and mitochondria by using Percoll to generate continuous density gradients. Both synaptosomal populations readily accumulated 14C-labelled alpha-ketoglutarate and L-malate by high-affinity transport systems. Hofstee plots of uptake velocity as a function of substrate concentration were highly nonlinear, indicating that uptake was mediated by two or more carriers, or was subject to negative cooperativity. At least one carrier was selective for alpha-ketoglutarate and another for malate, whereas a third carrier appeared to be present which transported both substrates. At low concentrations (approximately 1 microM), alpha-ketoglutarate transport was almost totally Na+-dependent, whereas malate uptake exhibited little Na+-dependency. The transport of alpha-ketoglutarate was associated with a net influx, and therefore was not due to a homoexchange process. alpha-Ketoglutarate and malate were metabolized rapidly to glutamate and aspartate, respectively, by both synaptosomal preparations; however, in all cases, label accumulated in gamma-aminobutyric acid rather slowly. The incorporation of label into glutamine from alpha-ketoglutarate was much greater in the high-density synaptosomes that in low-density synaptosomes, an indication that the former contained a higher proportion of astrogliasomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

A tandemly reiterated DNA sequence in the long repeat region of herpes simplex virus type 1 found in close proximity to immediate-early mRNA 1.

The 3' end of immediate-early mRNA 1 was mapped precisely within the IRL/TRL genome regions, and the DNA sequences around the 3' end were determined. An AATAAA polyadenylation signal was present 17 base pairs upstream of the 3' end, and eight tandemly repeated copies of a 16-base-pair sequence (GGGGGTGCGTGGGAGT) plus one further closely related copy were located 20 base pairs downstream. Other tandem reiterations present in the herpes simplex virus genome are described and their properties are considered.     

Intracellular Ca and cell injury: A paradoxical role of Ca in complement membrane attack

Disturbances in intracellular Ca2+ are known to be important in cell injury caused by a wide range of toxic factors. The complement system is a major effector of immune damage in vivo, and is known to be involved in the pathogenesis of many immune diseases. We present here evidence that the potentially lethal membrane attack complex of complement causes a rapid increase in intracellular free Ca2+ concentration before any other detectable biochemical changes in the cell. In nucleated cells the increased intracellular free Ca2+ concentration initially stimulates recovery processes, allowing the cell to escape mild complement attack and also activates the production of inflammatory mediators, which may amplify an ongoing inflammatory response. More severe complement membrane attack causes a more rapid rise in intracellular free Ca2+ concentration allowing a threshold to be breached above which recovery processes are overwhelmed, and cell death occurs. The importance of non-lytic effects and recovery processes mediated by Ca2+, and the molecular basis of these effects are discussed, and the hypothesis proposed that the cell-injuring effects of other "pore-forming" toxins are also caused by increases in intracellular free Ca2+.     

Column cellulose hydrolysis reactor: An efficient cellulose hydrolysis reactor with continuous cellulase recycling

Summary The use of a column cellulose hydrolysis reactor with continuous enzyme recycling was demonstrated by incorporating a continuous ultrafiltration apparatus at the effluent end of the column reactor. Using this setup, over 90% (w/v) cellulose hydrolysis was achieved, resulting in an average sugar concentration of 6.8% (w/v) in the effluent stream. The output of the system was 1.98 g of reducing sugar/l/h with a ratio of 87% (w/v) of the reducing sugars being monomeric sugars. Batch hydrolysis reactors were less effective, resulting in 57% (w/v) of the cellulose being hydrolyzed. The output of the batch reactor was 1.33 g of reducing sugar/l/h with similar product concentrations and percentage of monomeric sugars. The ratio of reducing sugar/filter paper unit of cellulase activity for the column method was 69.1 mg/U as compared to only 21.2 mg/U for the batch reactor.     

Structural basis of the polymorphism of human complement components C4A and C4B: gene size, reactivity and antigenicity.

The human complement components C4A and C4B are highly homologous proteins, but they show markedly different, class-specific, chemical reactivities. They also differ serologically in that C4A generally expresses the Rodgers (Rg) blood group antigens while C4B generally expresses the Chido (Ch) blood group antigens. C4A 1 and C4B 5 are exceptional variants which possess their class-specific chemical reactivities, but express essentially the reversed antigenicities. The genes encoding the typical Rg-positive C4A 3a and Ch-positive C4B 3 allotypes and the interesting variants C4A 1 and C4B 5 have been cloned. Characterization of the cloned DNA has revealed that the genes encoding the A 3a, A 1 and B 3 allotypes are 22 kb long, but that encoding B 5 is only 16 kb long. Comparison of derived amino acid sequences of the polymorphic C4d fragment has shown that C4A and C4B can be defined by only four isotypic amino acid differences at position 1101-1106. Over this region C4A has the sequence PCPVLD while C4B has the sequence LSPVIH, and this presumably is the cause of their different chemical reactivities. Moreover, the probable locations of the two Rg and the six Ch antigenic determinants have been deduced. Our structural data on the C4A and C4B polymorphism pattern suggests a gene conversion-like mechanism is operating in mixing the generally discrete serological phenotypes between C4A and C4B.     

Identification of the human papilloma virus-1a E4 gene products.

Antibodies prepared against a human papilloma virus-1 (HPV-1) E4/beta-galactosidase fusion protein identified several polypeptides in HPV-1, but not HPV-2 or 4, induced papillomas. The major E4 protein, that represented up to 30% of total cellular protein, was a 16/17-K doublet which was purified by column chromatography and analysed for amino acid content. A peptide derived by chymotryptic digestion was purified by h.p.l.c. and subjected to amino acid sequencing. The unique sequence obtained, Gly-His-Pro-Asp-Leu-Ser-Leu, identified the 16/17-K doublet as a product of the HPV-1 E4 gene region. Antibodies to both the E4/beta-galactosidase fusion protein and the 16/17-K doublet identified two smaller polypeptides (10/11-K) which may represent spliced products of E4. We propose that the products of the HPV-1 E4 gene region are not classical DNA tumor virus early proteins and suggest that they play a role in virus maturation.