A computer analysis of the amino acid sequences from the putative gene products of retroviral pol genes has revealed a 150-residue segment that is homologous with the ribonuclease H of Escherichia coli. The segment occurs at the carboxyl terminus of the region assigned to the 90-kDa reverse transcriptase polypeptide. In contrast, a section nearer the amino terminus of this sequence can be aligned with nonretroviral polymerases. (5), and an endonuclease ("integrase") that is essential for the integration of the newly synthesized DNA into the host genome (6).The pol gene of retroviruses is expressed initially as a gag-pol precursor that is proteolytically processed to a number of small gag proteins, an approximately 90-kDa protein encompassing both RNA-directed DNA polymerase (reverse transcriptase) and ribonuclease H activities, and, finally, a 40-kDa fragment with endonuclease activity (7). Several reports have presented evidence that the ribonuclease H activity of the 90-kDa reverse transcriptase portion is associated with the amino-terminal end of that protein, and by implication, that the DNA polymerase activity is at the carboxyl-terminal end. These conclusions are based on experiments involving deletion mutants (2), on the one hand, and antibodies to synthetic peptides modeled on the putative sequences, on the other (3).We now suggest that the opposite must be true: the ribonuclease H activity should be situated at the carboxyl terminus, and the DNA polymerase, at the amino terminus. We draw this conclusion on the basis of comparisons of the retroviral sequences with those of nonviral enzymes of similar function. In this regard, we have uncovered a significant resemblance between a 150-residue segment at the carboxyl-terminal end of the 90-kDa fragment and the reported sequence of a ribonuclease H from Escherichia coli. We also provide an alignment of a segment near the amino terminus of the 90-kDa polypeptide with highly conserved sequences from many other polymerases, including the a subunit of E. coli DNA-directed RNA polymerase. Finally, there is a distinctive sequence in the endonuclease sequence that is characteristic of a zinc-binding segment. METHODSThe sequences used were taken from the 1985 version of NEWAT (8) identical (Fig. 1). Binary comparison of each of the retroviral sequences with the E. coliribonuclease H sequence, followed by statistical evaluation by a randomization method, gave authentic alignment scores from 4 to 10 standard deviations above the means of the jumbled comparisons. The cumulative weight of the multiple alignment (Fig. 2) further bears out the significance of the overall relationship. That the polymerase portion of the viral reverse transcriptase system must encompass the amino-terminal portion of the 90-kDa fragment is established by the alignment shown in Fig. 3. The key region here involves a sector previously shown by Kamer and Argos (20) to be present in a number of nonretroviral polymerases; these consistently have two aspartic acid residues surrounded by...

The complete sequences of the RhsB and RhsC elements of Escherichia coli K-12 have been determined. These sequence data reveal a new repeated sequence, called H-rpt (Hinc repeat), which is distinct from the Rhs core repetition that is found in all five Rhs elements. H-rpt is found in RhsB, RhsC, and RhsE. Characterization of H-rpt supports the view that the Rhs elements are composite structures assembled from components with very different evolutionary histories and that their incorporation into the E. coli genome is relatively recent. In each case, H-rpt is found downstream from the Rhs core and is separated from the core by a segment of DNA that is unique to the individual element. The H-rpt's of RhsB and RhsE are very similar, diverging by only 2.1%. They are 1,291 bp in length, and each contains an 1,134-bp open reading frame (ORF). RhsC has three tandem copies of H-rpt, all of which appear defective in that they are large deletions and/or have the reading frame interrupted. Features of H-rpt are analogous to features typical of insertion sequences; however, no associated transposition activity has been detected. A 291-bp fragment of H-rpt is found near min 5 of the E. coli K-12 map and is not associated with any Rhs core homology. The complete core sequences of RhsB and RhsC have been compared with that of RhsA. As anticipated, the three core sequences are closely related, all having identical lengths of 3,714 bp each. Like RhsA, the RhsB and RhsC cores constitute single ORFs that begin with the first core base. In each case, the core ORF extends beyond the core into the unique sequence. Of the three cores, RhsB and RhsA are the most similar, showing only 0.9% sequence divergence, while RhsB and RhsC are the least similar, diverging by 2.9%. All three cores conserve the 28 repetitions of a peptide motif noted originally for RhsA. A secondary structure is proposed for this motif, and the possibility of its having an extracellular binding function is discussed. RhsB contains one additional unique ORF, and RhsC contains two additional unique ORFs. One of these ORFs includes a signal peptide that is functional when fused to TnphoA.

vir regulon expression in Agrobacterium tumefaciens involves both chromosome-and Ti-plasmid-encoded gene products. We (7,31,33,58

Cytokinin analysis by immunoaffinity chromatography (IAC), high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA) or enzyme-linked immunosorption assay (ELISA) has been used to study two separate topics: the role of tRNA in bacterial cytokinin biosynthesis and the changes in cytokinin concentration which occur during cereal grain development. Transfer RNA isopentenylation in the gall-forming plant pathogen Agrobacterium tumefaciens is encoded by the chromosomal miaA locus. Mutation of miaA reduces tRNA isopentenylation significantly and preliminary data suggest that turnover of isopentenylated tRNA is responsible for low level secretion of free N6-isopentenyladenine (iP) by the bacteria. However, the major route of cytokinin biosynthesis by gall-forming plant patho- genic bacteria is not via tRNA turnover but by direct biosynthesis mediated by dimethylallylpyro- phosphate: 5'-AMP transferase (DMAPP :AMP transferase) encoded by such genes as ipt, tzs (from A, tumefaciens) or ptz (from Pseudomonas savastanoi). Analysis of cytokinin levels in developing wheat and rice grains in the period immediately following pollination showed large transient increases in zeatin (Z) and zeatin riboside (ZR) which coincided with the period of maximum endosperm cell division reported by others. Detailed analyses of maize kernels, where development can be staged readily, showed that Z and ZR concentrations peaked 9 days after pollination (DAP). During the period 8-10 DAP, cytokinin oxidase underwent a significant increase in specific activity, indicating that cytokinin catabolism was enhanced as endosperm cell division ended.