Physical and genetic map of the major nif gene cluster from Azotobacter vinelandii.

Determination of a 28,793-base-pair DNA sequence of a region from the Azotobacter vinelandii genome that includes and flanks the nitrogenase structural gene region was completed. This information was used to revise the previously proposed organization of the major nif cluster. The major nif cluster from A. vinelandii encodes 15 nif-specific genes whose products bear significant structural identity to the corresponding nif-specific gene products from Klebsiella pneumoniae. These genes include nifH, nifD, nifK, nifT, nifY, nifE, nifN, nifX, nifU, nifS, nifV, nifW, nifZ, nifM, and nifF. Although there are significant spatial differences, the identified A. vinelandii nif-specific genes have the same sequential arrangement as the corresponding nif-specific genes from K. pneumoniae. Twelve other potential genes whose expression could be subject to nif-specific regulation were also found interspersed among the identified nif-specific genes. These potential genes do not encode products that are structurally related to the identified nif-specific gene products. Eleven potential nif-specific promoters were identified within the major nif cluster, and nine of these are preceded by an appropriate upstream activator sequence. A + T-rich regions were identified between 8 of the 11 proposed nif promoter sequences and their upstream activator sequences. Site-directed deletion-and-insertion mutagenesis was used to establish a genetic map of the major nif cluster.     

Characterization of the 5'' to 3'' exonuclease associated with Thermus aquaticus DNA polymerase.

Thermus aquaticus DNA polymerase was shown to contain an associated 5' to 3' exonuclease activity. Both polymerase and exonuclease activities cosedimented with a molecular weight of 72,000 during sucrose gradient centrifugation. Using a novel in situ activity gel procedure to simultaneously detect these two activities, we observed both DNA polymerase and exonuclease in a single band following either nondenaturing or denaturing polyacrylamide gel electrophoresis: therefore, DNA polymerase and exonuclease activities reside in the same polypeptide. As determined by SDS-polyacrylamide gel electrophoresis this enzyme has an apparent molecular weight of 92,000. The exonuclease requires a divalent cation (MgCl2 or MnCl2), has a pH optimum of 9.0 and excises primarily deoxyribonucleoside 5'-monophosphate from double-stranded DNA. Neither heat denatured DNA nor the free oligonucleotide (24-mer) were efficient substrates for exonuclease activity. The rate of hydrolysis of a 5'-phosphorylated oligonucleotide (24-mer) annealed to M13mp2 DNA was about twofold faster than the same substrate containing a 5'-hydroxylated residue. Hydrolysis of a 5'-terminal residue from a nick was preferred threefold over the same 5'-end of duplex DNA. The 5' to 3' exonuclease activity appeared to function coordinately with the DNA polymerase to facilitate a nick translational DNA synthesis reaction.     

Kinetics and location of bone marrow cell graft rejection by irradiated mice

Natural killer (NK) cells were eliminated with rabbit anti-Asialo GM1 (anti-ASGM1) serum to test the kinetics and location of bone marrow cell (BMC) rejection. Anti-ASGM1 serum was injected intravenously in mice at various times before or after irradiation (8.6 Gy) and transfer of parental-strain or allogeneic BMC. Growth of BMC was determined by measuring splenic 5-iodo-2'-deoxyuridine-125I incorporation 5 days after cell transfer. Anti-ASGM1 serum weakened hybrid resistance even if injected intravenously as late as 24 h post-BMC transfer and even in recipients injected with polyinosinic:polycytidylic acid so as to boost NK activity. If regenerating spleen cells (higher rate of cell cycling) were used as donor cells instead of BMC, the length of time required for rejection was unaffected. Anti-ASGM1 serum injected intravenously rapidly inhibited splenic NK activity and lung clearance of YAC-1 tumor cells, but when injected intratracheally, it only inhibited lung NK activity. Thus, BMC rejection occurs in the hematopoietic tissue and requires at least 24 h.     

Release of putative exocytic transport vesicles from perforated MDCK cells.

Mechanically perforated MDCK cells were used to study membrane transport between the trans-Golgi network and the apical and basolateral plasma membrane domains in vitro. Three membrane transport markers--an apical protein (fowl plague virus haemagglutinin), a basolateral protein (vesicular stomatitis virus G protein), and a lipid marker destined for both domains (C6-NBD-sphingomyelin)--were each accumulated in the trans-Golgi by a 20 degrees C block of transport and their behaviour monitored following cell perforation and incubation at 37 degrees C. In the presence of ATP and in the absence of calcium ions a considerable fraction of the transport markers were released from the perforated cells in sealed membrane vesicles. Control experiments showed that the vesicles were not generated by non-specific vesiculation of the Golgi complex or the plasma membrane. The vesicles had well defined sedimentation properties and the orientation expected of transport vesicles derived from the trans-Golgi network.     

Influence of blood parasites on the body mass of passeriform birds

Body masses of 3,739 birds representing immature and adult males and females of 15 species of passeriforms (both uninfected and infected with Haemoproteus spp. and Leucocytozoon spp.) were compared. There was some interaction among year, month and area of capture for several host species, but there was no discernible effect of either parasite genus on body mass. There were no effects due to high intensity parasitemia for eight host species examined. Either parasitism does not cause loss of body mass, or the techniques used were too insensitive to separate effects of parasitism from other natural causes.     

Microbial hydroxylation of quinoline in contaminated groundwater: evidence for incorporation of the oxygen atom of water

Studies conducted in an aquifer contaminated by creosote suggest that quinoline is converted to 2(1H)quinolinone by an indigenous consortium of microorganisms. Laboratory microbial experiments using H218O indicate that water is the source of the oxygen atom for this hydroxylation reaction under aerobic and anaerobic conditions.     

Social organization and ecology of proboscis monkeys (Nasalis larvatus) in mixed coastal forest in Sarawak

Data are presented from a 16-month study of proboscis monkeys in an area of mixed coastal forest in Sarawak. The population density, social organization, and feeding and ranging behavior are described in detail. Results are compared with those from other primates in an attempt to understand why females of certain species (including proboscis monkeys) transfer between social groups. The scarcity of available food and reasons for the limited habitat preferences of proboscis monkeys are also discussed.     

Mutations in human lymphocytes studied by an HLA selection system

Human lymphocytes mutated at the HLA-A2 or HLA-A3 alleles were enumerated and studied by primary selection using antibody and complement, followed by limiting dilution cloning and secondary selection using immunofluorescence or antibody and complement. The geometric mean frequency of in vivo mutant lymphocytes was 3.08 X 10(-5) for the HLA-A2 allele and 4.68 X 10(-6) for the HLA-A3 allele. Mutagenesis by X-radiation or mitomycin produced a dose-related increase in mutant frequency. HLA-B phenotyping and Southern Analysis of the HLA-A gene suggested that mutation was frequently due to gene deletion, which was often substantial.     

Sink Metabolism in Tomato Fruit : II. Phloem Unloading and Sugar Uptake

Analysis of [³H]-(fructosyl)-sucrose translocation in tomato (Lycopersicon esculentum Mill.) indicates that phloem unloading in the fruit occurs, at least in part, to the apoplast followed by extracellular hydrolysis. Apoplastic sucrose, glucose, and fructose concentrations were estimated as 1 to 7, 12 to 49, and 8 to 63 millimolar, respectively in the tomato fruit pericarp tissue. Hexose concentrations were at least four-fold greater than sucrose at all developmental stages. Short-term uptake of [¹⁴C]sucrose, -glucose, and -fructose in tomato pericarp disks showed first order kinetics over the physiologically relevant concentration range. The uptake rate of [¹⁴C]-(glucosyl)-1′-fluorosucrose was identical to the rate of [¹⁴C]sucrose uptake, suggesting sucrose may be taken up directly without prior extracellular hydrolysis. Short-term uptake of all three sugars was insensitive to 10 micromolar carbonyl cyanide m-chlorophenylhydrazone and to 10 micromolar p-chloromercuribenzene sulfonic acid. However, long-term accumulation of glucose was sensitive to carbonyl cyanide m-chlorophenylhydrazone. Together these results suggest that although sucrose is at least partially hydrolyzed in the apoplast, sucrose may enter the metabolic carbohydrate pool directly. In addition, sugar uptake across the plasma membrane does not appear to be energy dependent, suggesting that sugar accumulation in the tomato fruit is driven by subsequent intracellular metabolism and/or active uptake at the tonoplast.