Role of pit membranes in macromolecule-induced wilt of plants

Macromolecules present in low concentrations in xylem fluid of Medicago sativa L. var DuPuits will increase the resistance to xylem liquid flow. This increase in resistance was found to be reversible by backflushing the xylem. Autoradiography showed that very large molecules do not pass through pit membrane pores. A comparison of pit membrane pore sizes to molecule sizes suggests that increased resistance to xylem flow is a result of plugging pit membrane pores. It was also found that pit membranes located in two parts of the plant differ in the apparent diameter of their pores and, thus, in their susceptibility to plugging by macromolecules. Macromolecules in xylem fluid may result from hostparasite interactions and may play a significant role in the outcome of the interaction.     

Translation of poly(U) on ribosomes from Streptomyces aureofaciens

Slowly cooled cells of Streptomyces aureofaciens contained mainly tight-couple ribosomes. Maximum rate of polyphenylalanine synthesis on ribosomes of S. aureofaciens was observed at 40°C, while cultures grew optimally at 28°C. Ribosomes of S. aureofaciens differed from those of E. coli in the amount of poly(U) required for maximum synthetic activity. The polyphenylalanine-synthesizing activity of E. coli ribosomes was about 3-times higher than that of S. aureofaciens ribosomes. The addition of protein S1 of E. coli or the homologous protein from S. aureofaciens had no stimulatory effect on the translation of poly(U). In order to localize alteration(s) of S. aureofaciens ribosomes in the elongation step of polypeptide synthesis we developed an in vitro system derived from purified elongation factors and ribosomal subunits. The enzymatic binding of Phe-tRNA to ribosomes of S. aureofaciens was significantly lower than the binding to ribosomes of E. coli. This alteration was mainly connected with the function of S. aureofaciens 50 S subunits. These subunits were not deficient in their ability to associate with 30 S subunits or with protein SL5 which is homologous to L7/L12 of E. coli.     

Identification of sites of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen cross-linking in Escherichia coli 23S ribosomal ribonucleic acid

The reagent 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) was used to cross-link 23S rRNA from Escherichia coli under 50S ribosomal subunit reconstitution conditions. Following partial digestion of the RNA with ribonuclease T1, two-dimensional diagonal electrophoresis in denaturing polyacrylamide gels was used to isolate fragments derived from the cross-linked sites. These fragments were analyzed by digestion with ribonucleases T1 and A and their positions in the 23S RNA sequence identified. Fragment a1 (positions 1325-1426) is cross-linked to a2 (positions 1574-1623); fragment b1 (positions 1700-1731) is cross-linked to b2 (positions 1732-1753); and a cross-link is formed within fragment c (or c') (positions 863-916). In the latter case, the cross-link was located precisely, linking residues C867 and U913. All three HMT-mediated cross-links are consistent with a proposed secondary structure model for 23S RNA [Noller, H. F., Kop, J., Wheaton, V., Brosius, J., Gutell, R. R., Kopylov, A. M., Dohme, F., Herr, W., Stahl, D. A., Gupta, R., & Woese, C. R. (1981) Nucleic Acids Res. 9, 6167-6189].     

Unusual genome organization of the marine invertebrate Rapana thomasiana Grosse (Gastropoda)

The genome organization of the marine snail Rapana thomasiana Grosse (Gastropoda), genome size 2.7 pg, was studied by reassociation kinetics, S₁-nuclease assay, and restriction enzyme analysis. The slow-reassociating (single-copy) fraction represented only 21% of the genome. The average length of 80% of the single-copy sequences was less than 700 bp and the remaining 20% no longer than 1,400 bp. Longer stretches of unique DNA were not observed. The genome contained an unusually high percent-age of inverted repeats: at standard fragment length the zero-time binding fraction amounted to 25% of the genome. Foldback structures ranging from 200 bp to more than 10 kb were observed after S₁-nuclease treatment. They were randomly distributed throughout at least 85% of the genome, and the spacings between them were estimated to be about 1,600 bp on the average. The middle-repetitive DNA (45% of the genome) contained two kinetic components, repeated 430 and 65,000 times per genome, respectively. It was found that the majority of the repetitive sequences are about 300 bp long. Longer repeats (about 2,000 bp) were also observed, comprising a small portion of the genome. The inverted repeats, the middle-repetitive, and the singly-copy sequences were fully interspersed in the genome, thus indicating that R. thomasiana DNA is not organized in either the Xenopus or the Drosophila pattern type. — R. thomasiana is the only mollusc so far in which a satellite DNA has been found. It is organized in tandem repeats of 1,460 bp with a very complex organization but a low degree of divergence.     

Comparative analysis of the 5''-end regions of two repressible acid phosphatase genes in Saccharomyces cerevisiae.

The nucleotide sequence of 5'-noncoding and N-terminal coding regions of two coordinately regulated, repressible acid phosphatase genes from Saccharomyces cerevisiae were determined. These unlinked genes encode different, but structurally related polypeptides of molecular weights 60,000 and 56,000. The DNA sequences of their 5'-flanking regions show stretches of extensive homology upstream of, and surrounding, a "TATA" sequence and in a region in which heterogeneous 5' ends of the p60 mRNA were mapped. The predicted amino acid sequences encoded by the N-terminal regions of both genes were confirmed by determination of the amino acid sequence of the native exocellular acid phosphatase and the partial sequence of the presecretory polypeptide synthesized in a cell-free protein synthesizing system. The N-terminal region of the p60 polypeptide was shown to be characterized by a hydrophobic 17-amino acid signal polypeptide which is absent in the native exocellular protein and thought to be necessary for acid phosphatase secretion.     

Histone gene number and organisation in Xenopus: Xenopus borealis has a homogeneous major cluster

Using a Xenopus laevis H4 cDNA clone as a probe we have determined that the numbers of H4 histone genes in Xenopus laevis and Xenopus borealis are approximately the same. These numbers are dependent on the hybridization stringency and we measure about 90 H4 genes per haploid genome after a 60 degrees C wash in 3 X SSC. Using histone probes from both Xenopus and sea urchin we have studied the genomic organization of histone genes in these two species. In all of the X.borealis individuals analyzed about 70% of the histone genes were present in a very homogeneous major cluster. These genes are present in the order H1, H2B, H2A, H4 and H3, and the minimum length of the repeated unit is 16kb. In contrast, the histone gene clusters in X.laevis showed considerable sequence variation. However two major cluster types with different gene orders seem to be present in most individuals. The differences in histone gene organization seen in species of Xenopus suggest that even in closely related vertebrates the major histone gene clusters are quite fluid structures in evolutionary terms.     

The steady-state kinetics of isotope exchange at equilibrium: one substrate–one product enzymic mechanisms where two molecules of substrate or product are bound to an enzyme molecule (Short Communication)

The conclusion that the steady-state kinetics of isotope exchange at equilibrium do not show first-order behaviour for some one substrate-one product enzymic mechanisms in which two molecules of substrate or product can be combined with an enzyme molecule at the one time was shown to be erroneous.     

Production and utilization of acetate in mammals

1. In an attempt to define the importance of acetate as a metabolic precursor, the activities of acetyl-CoA synthetase (EC 6.2.1.1) and acetyl-CoA hydrolase (Ec 3.1.2.1) were assayed in tissues from rats and sheep. In addition, the concentrations of acetate in blood and liver were measured, as well as the rates of acetate production by tissue slices and mitochondrial fractions of these tissues. 2. Acetyl-CoA synthetase occurs at high activities in heart and kidney cortex of both species as well as in rat liver and the sheep masseter muscle. The enzyme is mostly in the cytosol fraction of liver, whereas it is associated with the mitochondrial fraction in heart tissue. Both mitochondrial and cytosol activities have a K(m) for acetate of 0.3mm. Acetyl-CoA synthetase activity in liver was not altered by changes in diet, age or alloxan-diabetes. 3. Acetyl-CoA hydrolase is widely distributed in rat and sheep tissues, the highest activity being found in liver. Essentially all of the activity in liver and heart is localized in the mitochondrial fraction. Hepatic acetyl-CoA hydrolase activity is increased by starvation in rats and sheep and during the suckling period in young rats. 4. The concentrations of acetate in blood are decreased by starvation and increased by alloxan-diabetes in both species. The uptake of acetate by the sheep hind limb is proportional to the arterial concentration of acetate, except in alloxan-treated animals, where uptake is impaired. 5. Acetate is produced by liver and heart slices and also by heart mitochondrial fractions that are incubated with either pyruvate or palmitoyl-(-)-carnitine. Liver mitochondrial fractions do not form acetate from either substrate but instead convert acetate into acetoacetate. 6. We propose that acetate in the blood of rats or starved sheep is derived from the hydrolysis of acetyl-CoA. Release of acetate from tissues would occur under conditions when the function of the tricarboxylic acid cycle is restricted, so that the circulating acetate serves to redistribute oxidizable substrate throughout the body. This function is analogous to that served by ketone bodies.