Functional analysis of Arg-308 mutants of Flp recombinase. Possible role of Arg-308 in coupling substrate binding to catalysis

The arginine residue at position 308 in the Flp recombinase corresponds to the only invariant arginine within the Int family of recombinases. Alterations of this residue result in Flp variants that retain substrate recognition, but form weaker protein-DNA complexes than wild type Flp. Furthermore, their DNA cleavage activity is significantly diminished. A conservative change of R308K results in a functional Flp variant; however, this protein has a lowered temperature optimum for recombination. The Arg-308 mutants can be stabilized on the DNA substrate through cooperativity with a partner Flp mutant that is tight binding. Thus, interactions between Flp monomers must be a relevant feature of the normal recombination reaction.     

The Sabinas syndrome

We have defined a new autosomal recessive disorder in patients stemming from a small community in northern Mexico. Diagnosable at birth, its major symptoms include brittle hair, mental retardation, and nail dysplasia. Structural hair abnormalities are seen by both light and electron microscopy. Hair cystine content is reduced while the copper/zinc ratio in hair is increased.     

Induction of Flavonoid Synthesizing Enzymes by Light in Etiolated Pea (Pisum sativum cv. Midfreezer) Seedlings

Etiolated pea (Pisum sativum cv. Midfreezer) seedlings respond to illumination with white light by changes in the activity of phenylpropanoid and flavonoid synthesizing enzymes. Unlike in cell cultures, changes in enzyme activity in pea seedlings are not concerted. Phenylalanine ammonia-lyase (EC 4.3.1.5) activity peaked approximately 18 hours after onset of illumination. The phenylacetate path did not interfere with the measurement of phenylalanine ammonia-lyase activity. Activity of cinnamic acid 4-hydroxylase (EC 1.14.13.11) showed an early peak after 8 hours illumination, declined thereafter sharply, then gradually increased during the remainder of the experiment. Activities of chalcone synthase and UDP glucose:flavonol 3-O-glucosyltransferase (EC 2.4.1.91) increased steadily and reached a plateau after approximately 70 hours illumination time. Activity of 4-hydroxycinnamate:coenzyme A ligase (EC 6.2.1.12) remained relatively unchanged, whereas that of chalcone isomerase (EC 5.5.1.6) declined steadily during the course of the experiment. The relative in vitro enzyme activities suggest that the rate-limiting step for the phenylpropanoid path is the cinnamic acid 4-hydroxylase, that of the flavonoid pathway is the chalcone synthase. Integration of enzyme activity curves, however, show that only the curve deriving from phenylanine ammonia-lyase activity matches closely the production of the flavonol glycosides.     

Avian Retrovirus pp32 DNA-Binding Protein I. Recognition of Specific Sequences on Retrovirus DNA Terminal Repeats

The avian retrovirus pp32 protein possesses a DNA-nicking activity which prefers supercoiled DNA as substrate. We have investigated the binding of pp32 to avian retrovirus long terminal repeat (LTR) DNA present in both supercoiled and linear forms. The cloned viral DNA was derived from unintegrated Schmidt-Ruppin A (SRA) DNA. A subclone of the viral DNA in pBR322 (termed pPvuII-DG) contains some src sequences, tandem copies of LTR sequences, and partial gag sequences in the order src-U(3) U(5):U(3) U(5)-gag. Binding of pp32 to supercoiled pPvuII-DG DNA followed by digestion of this complex with a multicut restriction enzyme (28 fragments total) permitted pp32 to preferentially retain on nitrocellulose filters two viral DNA fragments containing only LTR DNA sequences. In addition, pp32 also preferentially retained four plasmid DNA fragments containing either potential promoters or Tn3 "left-end" inverted repeat sequences. Mapping of the pp32 binding sites on viral LTR DNA was accomplished by using the DNase I footprinting technique. The pp32 protein, but not the avian retrovirus alphabeta DNA polymerase, is able to form a unique protein-DNA complex with selected regions of either SRA or Prague A LTR DNAs. Partial DNase I digestion of a 275-base pair SRA DNA fragment complexed with pp32 gives upon electrophoresis in denaturing gels a unique ladder pattern, with regions of diminished DNase I susceptibility from 6 to 10 nucleotides in length, in comparison with control digests in the absence of protein. The binding of pp32 to this fragment also yields enhanced DNase I-susceptible sites that are spaced between the areas protected from DNase I digestion. The protected region of this unique complex was a stretch of 170 +/- 10 nucleotides that encompasses the presumed viral promoter site in U(3), which is adjacent to the src region, extends through U(5), and proceeds past the joint into U(3) for about 34 base pairs. No specific protection or DNase I enhancement by pp32 was observed in experiments with a 435-base pair SRA DNA fragment derived from a part of U(3) and the adjacent src region or a 55-base pair DNA fragment derived from another part of U(3). The DNA sequence of Prague A DNA at the fused LTRs differs from that of SRA DNA. The alteration in the sequence at the juncture of the LTRs prevented pp32 from forming a stable complex in this region of the LTR. Our results are relevant to two aspects of the interaction between pp32 and LTR DNA. First, the pp32 protein in the presence of selected viral DNA restriction fragments possibly forms a higher order oligomer analogous to Escherichia coli DNA gyrase-DNA complexes or eucaryotic nucleosome structures. Second, the specificity of the binding suggests a role for pp32 and the protected DNA sequences in the retrovirus life cycle. The preferred sequences to which pp32 binds include two adjacent 15-base pair inverted terminal repeats at the joint between U(5) and U(3) in SRA DNA. This region is involved in circularization of linear DNA and is perhaps the site that directs integration into cellular DNA.     

Arrangement of Integrated Avian Sarcoma Virus DNA Sequences Within the Cellular Genomes of Transformed and Revertant Mammalian Cells

We have examined the arrangement of integrated avian sarcoma virus (ASV) DNA sequences in several different avian sarcoma virus transformed mammalian cell lines, in independently isolated clones of avian sarcoma virus transformed rat liver cells, and in morphologically normal revertants of avian sarcoma virus transformed rat embryo cells. By using restriction endonuclease digestion, agarose gel electrophoresis, Southern blotting, and hybridization with labeled avian sarcoma virus complementary DNA probes, we have compared the restriction enzyme cleavage maps of integrated viral DNA and adjacent cellular DNA sequences in four different mouse and rat cell lines transformed with either Bratislava 77 or Schmidt-Ruppin strains of avian sarcoma virus. The results of these experiments indicated that the integrated viral DNA resided at a different site within the host cell genome in each transformed cell line. A similar analysis of several independently derived clones of Schmidt-Ruppin transformed rat liver cells also revealed that each clone contained a unique cellular site for the integration of proviral DNA. Examination of several morphologically normal revertants and spontaneous retransformants of Schmidt-Ruppin transformed rat embryo cells revealed that the internal arrangement and cellular integration site of viral DNA sequences was identical with that of the transformed parent cell line. The loss of the transformed phenotype in these revertant cell lines, therefore, does not appear to be the result of rearrangement or deletions either within the viral genome or in adjacent cellular DNA sequences. The data presented support a model for ASV proviral DNA integration in which recombination can occur at multiple sites within the mammalian cell genome. The integration and maintenance of at least one complete copy of the viral genome appear to be required for continuous expression of the transformed phenotype in mammalian cells.     

Purification and characterization of cytochrome c oxidase from rat liver mitochondria.

Cytochrome c oxidase has been purified from rat liver mitochondria using affinity chromatography. The preparation contains 10.5 to 13.4 nmol of heme a + a3 per mg of protein and migrates as a single band during polyacrylamide gel electrophoresis under nondissociating conditions. It has a heme a/a3 ratio of 1.12 and is free of cytochromes b, c, and c1 as well as the enzymes, NADH dehydrogenase, succinic dehydrogenase, coenzyme Q-cytochrome c reductase, and ATPase. The enzyme preparation consists of six polypeptides having apparent Mr of 66,000, 39,000, 23,000, 14,000, 12,500 and 10,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The peptide composition is similar to those found for cytochrome c oxidases from other systems. The enzymatic activity of the purified enzyme is completely inhibited by carbon monoxide or cyanide, partially inhibited by Triton X-100 and dramatically enhanced by Tween 80 or phospholipids.     

Mixing an enclosed, 1300m3 water column: effects on the planktonic food web

The nutrient, phytoplankton, and zooplankton dynamics in threeenclosed water columns (1300 m³) are described. Two of the enclosureswere mixed using a bubbling chamber at depth. Young chum salmon(Oncorhynchus keta) were added to one of the mixed enclosuresand the unmixed enclosure. No other manipulations were imposed.Copepods appeared in large numbers (e.g. especially Pseudocalanusminutus s.l. and Paracalanus parvus) and population growth rateswere estimated. Ctenophora did not appear in large numbers despitepresumably ideal food environments; it is suggested that inone enclosure this is a consequence of fish predation on thectenophores. The fish experienced high mortalities and low growthrates presumably due to unsuitable prey size. Weekly collectionsof sediment permitted isolation of two major sediment contributors,the first from phytoplankton sinking and the second from biogenkfallout associated with herbivore production. It was found thatthe more oligotrophic enclosure (unmixed) experienced proportionallyhigher utilization of organic carbon. Some of these resultsare explained by our data while others require more sophisticatedexperimentation, both in the design of the containers and inthe types of observations.     

Biosynthetic direction substrate kinetics and product inhibition studies on the first enzyme of histidine biosynthesis, adenosine triphosphate phosphoribosyltransferase.

Initial velocity steady-state substrate kinetics for the ATP phosphoribosyltransferase reaction in the biosynthetic direction were determined and are consistent with a sequential kinetic mechanism. To hold the fractions of magnesium-complexed substrates and products constant so as to avoid possible distortion of reciprocal velocity plots Mg²⁺ binding constants to the substrates ATP and phosphoribosylpyrophosphate and the product pyrophosphate were measured under assay conditions. Several conformational states of the phosphoribosyltransferase distinguishable by other criteria gave similar substrate kinetic behavior. Product inhibition studies were conducted to elucidate the binding order. Phosphoribosyl-ATP was competitive with respect to ATP and was non-competitive with respect to phosphoribosylpyrophosphate. Pyrophosphate was non-competitive with respect to both substrates. The data are consistent with the ordered Bi-Bi kinetic mechanism with ATP binding first to free enzyme and phosphoribosyl-ATP dissociating last from enzyme-product complexes.