The role of CDC28 and cyclins during mitosis in the budding yeast S. cerevisiae.

cdc28-1N is a conditional allele that has normal G1 (Start) function but confers a mitotic defect. We have isolated seven genes that in high dosage suppress the growth defect of cdc28-1N cells but not of Start-defective cdc28-4 cells. Three of these (CLB1, CLB2, and CLB4) encode proteins strongly homologous to G2-specific B-type cyclins. Another gene, CLB3, was cloned using PCR, CLB1 and CLB2 encode a pair of closely related proteins; CLB3 and CLB4 encode a second pair. Neither CLB1 nor CLB2 is essential; however, disruption of both is lethal and causes a mitotic defect. Furthermore, the double mutant cdc28-1N clb2::LEU2 is nonviable, whereas cdc28-4 clb2::LEU2 is viable, suggesting that the cdc28-1N protein may be defective in its interaction with B-type cyclins. Our results are consistent with CDC28 function being required in both G1 and mitosis. Its mitotic role, we believe, involves interaction with a family of at least four G2-specific cyclins.     

Characterization of de novo duplications in eight patients by using fluorescence in situ hybridization with chromosome-specific DNA libraries.

Fluorescence in situ hybridization (FISH) with chromosome-specific DNA libraries was performed on samples from eight patients with de novo chromosomal duplications. In five cases, the clinical phenotype and/or cytogenetic evaluations suggested a likely origin of the duplicated material. In the remaining three cases, careful examination of the GTG-banding pattern indicated multiple possible origins; hybridization with more than one chromosome-specific library was performed on two of these cases. In all cases, FISH conclusively identified the chromosomal origin of the duplicated material. In addition, the hybridization pattern was useful in quantitatively delineating the duplication in two cases.     

Purification and characterization of catalase from goat (Capra capra) lung

Catalase plays a major role in the protection of tissues from toxic effects of H₂O₂ and partially reduced oxygen species. In the present study catalase was extracted and purified 330-fold from goat lung by acetone fractionation and successive chromatographies on DEAE-cellulose, Sephadex G-200, Blue Sepharose CL-6B and Ultrogel AcA-34. The purified enzyme was almost homogeneous as judged by polyacrylamide gel electrophoresis and FPLC. The molecular weight and Stokes' radius of the purified enzyme were 339 kDa and 127±2 Å. The enzyme had 11 sulfhydryl groups and 15 tryptophan groups per mol of the enzyme. A broad pH optimum in the range 5.2 to 7.8 was obtained. Sulfhydryl group binding agents, thiol reagents and N-Bromosuccinimide inhibited the enzyme activity. The kinetic data show no cooperativity between the substrate binding sites. Tryptophan, indole acetic acid, cysteine, formaldehyde and sodium azide inhibited the enzyme non-competitively with K_i values of 1.5, 1.6, 6.7, 0.55 and 0.0017 mM, respectively.     

Initiation of Protein Synthesis Without Formylation in a Mutant of Escherichia coli That Grows in the Absence of Tetrahydrofolate

Starting from a p-aminobenzoate-requiring strain of Escherichia coli (E. coli K-12 AB3292), we have isolated mutants that can grow in the absence of p-aminobenzoate (and thus tetrahydrofolate). The following lines of evidence suggest that at least one of these mutants is capable of initiating protein synthesis without formylation of methionyl-transfer ribonucleic acid (methionyl-tRNA(fMet)). (i) tRNA isolated (and charged in vivo with [(35)S]methionine) from this mutant grown in a p-aminobenzoate-free medium contained less than 0.4% of the total methionine charged to the tRNA as formylmethionine. However, when the mutant was grown in the presence of p-aminobenzoate, 40 to 50% of the total [(35)S]methionine was detected as formylmethionine. (ii) Extracts of the mutant grown in the absence of p-aminobenzoate contained no formyl-tetrahydrofolate, but such extracts did contain formylatable methionyl-tRNA and a functional transformylase. (iii) Tetrahydrofolate-free extracts of the mutant were capable of supporting protein synthesis with viral RNA (from f2) as messenger, but the resulting synthesized proteins contained no formylmethionine, and methionine residues were detected where formylmethionine residues are normally found. In the presence of formyl-tetrahydrofolate, use of a similar extract resulted in the detection of 30 to 40% of the total polypeptide methionine as formylmethionine. (iv) Initiation of protein synthesis in vitro occurred more readily with formyl-tetrahydrofolate-free extracts of the mutant than with similar extracts prepared from the parent strain. However, in the presence of formyl-tetrahydrofolate, initiation of protein synthesis proceeded equally well with both kinds of extracts. tRNA from this mutant and another spontaneously derived mutant was found to be partially deficient in the modified nucleoside ribothymidine (rT). Analysis of extracts showed that the mutants contained decreased levels of the methylase that results in the formation of ribothymidine. In vivo studies with an independently isolated rT(-) strain suggest that the lack of rT in tRNA facilitates the growth of E. coli under conditions where protein synthesis is forced to take place without formylation.     

Secondary structure of mouse and rabbit α- and β-globin mRNAs: Differential accessibility of α and β initiator AUG codons towards nucleases

The nucleotide sequence from the 5′ terminus inward of one third of mouse α- and β^maj-globin messenger RNAs has been established. In addition, using 5′ ³²P end-labeled mRNAs as substrates and S1 and T1 nucleases as probes for single-stranded regions, the secondary structures of mouse and rabbit α- and β-globin mRNAs have been analyzed. Our results indicate that the AUG initiator codon in both mouse and rabbit β-globin mRNA is quite susceptible to cleavage with S1 and T1 nucleases, suggesting that it resides in a single-stranded exposed region. In contrast, the initiator AUG in the α-globin mRNA of both species is inaccessible to cleavage, indicating that it is either buried by tertiary structure or is base-paired. Since the rate of initiation of protein synthesis with β-globin mRNA in rabbit reticulocyte is 30–40% faster than for α-globin mRNA, these results imply a possible correlation between the differential rates of initiation with these two mRNAs and the accessibility of the respective AUG initiator codons.     

Glucose transporter isotypes switch in T-antigen-transformed pancreatic beta cells growing in culture and in mice.

High-level expression of the low-Km glucose transporter isoform GLUT-1 is characteristic of many cultured tumor and oncogene-transformed cells. In this study, we tested whether induction of GLUT-1 occurs in tumors in vivo. Normal mouse beta islet cells express the high-Km (approximately 20 mM) glucose transporter isoform GLUT-2 but not the low-Km (1 to 3 mM) GLUT-1. In contrast, a beta cell line derived from an insulinoma arising in a transgenic mouse harboring an insulin-promoted simian virus 40 T-antigen oncogene (beta TC3) expressed very low levels of GLUT-2 but high levels of GLUT-1. GLUT-1 protein was not detectable on the plasma membrane of islets or tumors of the transgenic mice but was induced in high amounts when the tumor-derived beta TC3 cells were grown in tissue culture. GLUT-1 expression in secondary tumors formed after injection of beta TC3 cells into mice was reduced. Thus, high-level expression of GLUT-1 in these tumor cells is characteristic of culture conditions and is not induced by the oncogenic transformation; indeed, overnight culture of normal pancreatic islets causes induction of GLUT-1. We also investigated the relationship between expression of the different glucose transporter isoforms by islet and tumor cells and induction of insulin secretion by glucose. Prehyperplastic transgenic islet cells that expressed normal levels of GLUT-2 and no detectable GLUT-1 exhibited an increased sensitivity to glucose, as evidenced by maximal insulin secretion at lower glucose concentrations, compared with that exhibited by normal islets. Further, hyperplastic islets and primary and secondary tumors expressed low levels of GLUT-2 and no detectable GLUT-1 on the plasma membrane; these cells exhibited high basal insulin secretion and responded poorly to an increase in extracellular glucose. Thus, abnormal glucose-induced secretion of insulin in prehyperplastic islets in mice was independent of changes in GLUT-2 expression and did not require induction of GLUT-1 expression.     

Predicting the distributions of predator (snow leopard) and prey (blue sheep) under climate change in the Himalaya

Future climate change is likely to affect distributions of species, disrupt biotic interactions, and cause spatial incongruity of predator–prey habitats. Understanding the impacts of future climate change on species distribution will help in the formulation of conservation policies to reduce the risks of future biodiversity losses. Using a species distribution modeling approach by MaxEnt, we modeled current and future distributions of snow leopard (Panthera uncia) and its common prey, blue sheep (Pseudois nayaur), and observed the changes in niche overlap in the Nepal Himalaya. Annual mean temperature is the major climatic factor responsible for the snow leopard and blue sheep distributions in the energy‐deficient environments of high altitudes. Currently, about 15.32% and 15.93% area of the Nepal Himalaya are suitable for snow leopard and blue sheep habitats, respectively. The bioclimatic models show that the current suitable habitats of both snow leopard and blue sheep will be reduced under future climate change. The predicted suitable habitat of the snow leopard is decreased when blue sheep habitats is incorporated in the model. Our climate‐only model shows that only 11.64% (17,190 km²) area of Nepal is suitable for the snow leopard under current climate and the suitable habitat reduces to 5,435 km² (reduced by 24.02%) after incorporating the predicted distribution of blue sheep. The predicted distribution of snow leopard reduces by 14.57% in 2030 and by 21.57% in 2050 when the predicted distribution of blue sheep is included as compared to 1.98% reduction in 2030 and 3.80% reduction in 2050 based on the climate‐only model. It is predicted that future climate may alter the predator–prey spatial interaction inducing a lower degree of overlap and a higher degree of mismatch between snow leopard and blue sheep niches. This suggests increased energetic costs of finding preferred prey for snow leopards – a species already facing energetic constraints due to the limited dietary resources in its alpine habitat. Our findings provide valuable information for extension of protected areas in future.     

Establishment of mammalian cell lines containing multiple nonsense mutations and functional suppressor tRNA genes

We describe the generation of mammalian cell lines carrying amber suppressor genes. Nonsense mutants in the herpes simplex virus thymidine kinase (HSV tk) gene, the Escherichia coli xanthine-guanine phosphoribosyl transferase (Eco-gpt) gene and the aminoglycoside 3′ phosphotransferase gene of the Tn5 transposon (NPT-II) were isolated and characterized. Each gene was engineered with the appropriate control signals to allow expression in both E. coli and mammalian cells. Expression in E. coli made possible the use of well developed bacterial and phage genetic manipulations to isolate and characterize the nonsense mutants. Once characterized, the nonsense mutants were transferred into mammalian cells by microinjection and used, in turn, to select for amber suppressor genes. Xenopus laevis amber suppressor genes, prepared by site-specific mutagenesis of a normal X. laevis tRNA gene, were microinjected into the above cell lines and selected for the expression of one or more of the amber mutant gene products. The resulting cell lines, containing functional amber suppressor genes, are stable and exhibit normal growth rates.     

Detection of antimycin-binding subunits of Complex III by photoaffinity-labeling with an azido derivative of antimycin

Deformamidoazidoantimycin A (DAA), a photoactive derivative of antimycin A containing an azido group substituting for the formamido group attached to the phenyl ring, was synthesized. The ultraviolet spectrum of DAA was almost identical to that of antimycin A, indicating little alteration of the electronic structure of the substituted phenyl ring by the azido substitution. However, the inhibitory effectiveness of DAA toward ubiquinol-cytochromec reductase (Complex III) purified from bovine heart (K _i =ca. 0.5 µM) was considerably less than that of antimycin (K _i 3 pM), indicating a direct rather than a supporting role of the formamido group in the inhibitory activity of antimycin. Exposure of purified Complex III to [³H]DAA plus ultraviolet light caused a major labeling by tritium of SDS-PAGE band 7 (m=13 kDa by SDS-PAGE) and lesser but significant labeling of bands 3, 6, 8, and 9. Pretreatment of Complex III with antimycin greatly suppressed the labeling of bands 5, 6, and 7 but caused an apparent increased labeling of bands 8 and 9 by [³H]DAA, respectively. The labeling of band 7 by [³H]DAA also was strongly suppressed by reduction of Complex III by either sodium borohybride or ascorbate. Based on magnitude of labeling by [³H]DAA and the degree of suppression of labeling by antimycin, the protein of band 7 qualified as the principal component for specific binding of antimycin with the protein of band 6 (m=16 kDa) showing a lesser but significant amount of specific binding.