The 5S rRNA gene in sea barley (Hordeum marinum Hudson sensu lato): sequence variation among repeat units and relationship to the X haplome in barley (Hordeum)

We have investigated the molecular diversity of the 5S rDNA units in sea barley, comprising Hordeum marinum and Hordeum geniculatum. Although we were unable to detect "short" units after screening of 639 clones, we found two unit classes, one 602-607 bp long and the other 507-512 bp long. We classify the shortest unit class of the two as belonging to the "long H1" unit class, identified in previous papers. The longest unit class is not similar to any unit class so far identified, and is therefore unique. It was coined by us as the "long X1," to reflect the X haplome. We present a summary of all the unit classes so far described in Hordeum. We carried out a cladistic analysis, based on the "long H1" (orthologous) sequences, that included H. vulgare, H. spontaneum, H. bulbosum, H. marinum, H. geniculatum, and H. bogdanii. As a result, the first three grouped in one clade, and the other three in the other clade, with the latter clade being more isolated. These results reflect current knowledge of relationships based on morphology, cytology, and genome analysis. Furthermore, the sequences from the 5S unit classes may be potentially useful as DNA probes for genomic identification and genetic transfer in the Triticeae.     

Variation of nitrate reductase genes in selected grass species

In order to study the variation of nitrate reductase (NR) genes among grass species, gene number, intron size and number, and the heme-hinge fragment sequence of 25 grass species were compared. Genomic DNA cut with six restriction enzymes and hybridized with the barley NAD(P)H and NADH NR gene probes revealed a single NAD(P)H NR gene copy and two or more NADH NR gene copies per haploid genome in most of the species examined. Major exceptions were Hordeum vulgare, H. vulgare ssp. spontaneum, and Avena strigosa, which appeared to have a single NADH NR gene copy. The NADH NR gene intron number and lengths were examined by polymerase chain reaction amplification. Introns I and III appeared to be absent in at least one of the NADH NR genes in the grass species, while intron II varied from 0.8 to 2.4 kilobases in length. The NADH NR gene heme-hinge regions were amplified and sequenced. The estimated average overall nucleotide substitution rate in the sequenced region was 7.8 x 10(-10) substitutions/site per year. The synonymous substitution rate was 2.11 x 10(-9) substitutions/synonymous site per year and the nonsynonymous substitution rate was 4.10 x 10(-10) substitutions/nonsynonymous site per year. Phylogenetic analyses showed that all of the wild Hordeum species examined clustered in a group separate from H. vulgare and H. vulgare ssp. spontaneum.     

DNA repair enzyme uracil DNA glycosylase is specifically incorporated into human immunodeficiency virus type 1 viral particles through a Vpr-independent mechanism

The Vpr protein, encoded by the human immunodeficiency virus type 1 (HIV-1) genome, is one of the nonstructural proteins packaged in large amounts into viral particles. We have previously reported that Vpr associates with the DNA repair enzyme uracil DNA glycosylase (UDG). In this study, we extended these observations by investigating whether UDG is incorporated into virions and whether this incorporation requires the presence of Vpr. Our results, with highly purified viruses, show that UDG is efficiently incorporated either into wild-type virions or into Vpr-deficient HIV-1 virions, indicating that Vpr is not involved in UDG packaging. Using an in vitro protein-protein binding assay, we reveal a direct interaction between the precursor form of UDG and the viral integrase (IN). Finally, we demonstrate that IN-defective viruses fail to incorporate UDG, indicating that IN is required for packaging of UDG into virions.     

Nucleotide sequence of the murine leukaemia virus amphotropic strain 4070A integrase (IN) coding region and comparative structural analysis of the inferred polypeptide

The complete nucleotide sequence of the integrase (IN) protein coding region of the murine leukaemia virus (MLV) amphotropic strain 4070A is presented. The sequence comprises 1,224 nucleotides, encoding a 408-residue polypeptide of M(r) 46,312. Alignment of the inferred 4070A IN amino acid sequence with the IN proteins of other MLV showed that substitutions are confined largely to segments within the N- and C-terminal domains. In the N-terminal domain the majority of substitutions occur as contiguous 2- to 6-residue blocks, whereas in the C-terminal domain they occur as isolated entities except within a short segment characterized by deletions/insertions. Selection appears to act on the C-terminal 19 residues of IN rather than on the N-terminal residues of ENV (encoded by overlapping reading frames), suggesting a functional role for this segment. Phylogenetic analyses grouped the sequences into two clusters, one comprising IN from the amphotropic strain 4070A and three ecotropic MLV (CAS-BR-E, Moloney and Friend), the other consisting of IN from three ecotropic MLV (two radiation-induced viruses and AKV) and a mink cell focus-forming (MCF) MLV virus. The same dichotomy and cluster composition was obtained from analysis of the long terminal repeat (LTR) regions from these viruses (consistent with the functional interrelationship of IN and LTR) but not from analysis of envelope protein sequences (consistent with the functional independence of ENV proteins from both IN and LTR). Secondary structure predictions supported features determined from the catalytic domain of human immunodeficiency virus and avian sarcoma virus IN, and identified probable structures within the relatively long N- and C-terminal domains of MLV IN proteins.     

Cryo-electron microscopy structure of yeast Ty retrotransposon virus-like particles

The virus-like particles (VLPs) produced by the yeast retrotransposon Ty1 are functionally related to retroviral cores. These particles are unusual in that they have variable radif. A paired mass-radius analysis of VLPs by scanning transmission electron microscopy showed that many of these particles form an icosahedral T-number series. Three-dimensional reconstruction to 38-A resolution from cryo-electron micrographs of T = 3 and T = 4 shells revealed that the single structural protein encoded by the TYA gene assembles into spiky shells from trimeric units.     

Behavioral factors affecting exposure potential for household cleaning products

Variation in repeated nucleotide sequences (RNSs) at the level of entire families assayed by Southern blot hybridization is remarkably low within species and is a powerful tool for scrutinizing the origin of allopolyploid taxa. Thirty-one clones from RNSs isolated from different Triticeae genera were used to investigate the genome constitution of South American Elymus. One of these clones, pHch2, preferentially hybridized with the diploid H genome Hordeum species. Hybridization of this clone with a worldwide collection of Elymus species with known genome formulas showed that pHch2 clearly discriminates Elymus species with the H genome (StH, StHH, StStH, and StHY) from those with other genome combinations (StY, StStY, StPY, and StP). Hybridization with pHch2 indicates the presence of the H genome in all South American Elymus species except Elymus erianthus and Elymus mendocinus. Hybridization with additional clones that revealed differential restriction fragments (marker bands) for the H genome confirmed the absence of the H genome in these species. Differential restriction fragments for the Ns genome of Psathyrostachys were detected in E. erianthus and E. mendocinus and three species of Leymus. Based on genome constitution, morphology, and habitat, E. erianthus and E. mendocinus were transferred to the genus Leymus.     

Nucleotide sequence of the genome of a citrus isolate of olive latent virus 1

The 3699 nt genome of olive latent virus 1 (OLV-1), described years ago from Southern Italy as a putative sobemovirus, was completely sequenced. OLV-1 genomic RNA was not polyadenylated and had a structure virtually identical to that of species of the Necrovirus rather than the Sobemovirus genus. Five open reading frames (ORFs) were identified, of which the 5'-proximal encoded a 23K protein and ended with an amber codon whose readthrough could yield a putative 82K product. This polypeptide had extensive sequence similarity with polymerases of serotypes A and D of tobacco necrosis necrovirus (TNV-A and TNV-D) and species of the family Tombusviridae and related genera (Dianthovirus and Machlomovirus). Two small ORFs followed, which encoded polypeptides of 8K and 6K, respectively. The 6K product had extensive homology with the comparable 6K protein of TNV-A and was also related to the 11K protein of shallot latent carlavirus, one of the "triple block" polypeptides involved in cell-to-cell virus movement. The 3'-proximal ORF was in the same position as the coat protein (CP) cistron of necroviruses and encoded a 30K product related to CP of both TNV-A and -D. Computer-assisted comparative analysis of structural and non-structural proteins of OLV-1, TNV-A and TNV-D disclosed on overall distant relationship between OLV-1 and TNV-D. OLV-1 genome appeared homologous to that of TNV-A, but differences from TNV-A were the absence of the small ORF downstream of the CP cistron and in the low degree of sequence identity in CP (39% aa identity). OLV-1 is serologically distantly related to TNV-A and even more distantly related to TNV-D. We propose that OLV-1 is a necrovirus species in its own right.     

Repeated DNA sequences isolated by microdissection. I. Karyotyping of barley (Hordeum vulgare L.)

We report on microdissection, cloning and sequence, and Southern and fluorescence in situ hybridization (FISH) analysis of one moderately and one highly amplified repetitive DNA element, pHvMWG2314 and pHvMWG2315, respectively, isolated from barley (Hordeum vulgare L.) chromosome arm 3HL. The pHvMWG2315 sequence hybridizes to all 14 telomeric or subtelomeric regions of the barley chromosomes as determined by FISH. The 50 different hybridization sites that include intercalary signals allow the discrimination of all 14 chromosome arms and the construction of a kariotype of barley. The tandemly repeated subtelomeric element of 331 bp exists in all Triticeae species tested (H. vulgare, Agropyron elongatum, Secale cereale, Triticum tauschii, T. turgidum, and T. aestivum). It is AT rich (66%), exibits 84% sequence homology to subfragments of the D genome ?specific? 1-kb element pAs1 of T. tauscii and 75% homology to interspersed genome-specific DNA sequence pHcKB6 from H. chilence. The repetitive sequence pHvMWG2314 is moderately amplified in barley and highly amplified in hexaploid wheat. The in situ experiments revealed no distinct signals on barley chromosomes, indicating a dispersed character for the sequence. The significance of the results for the identification of chromosomes and chromosome aberrations in FISH experiments are discussed.     

Mutations in the human immunodeficiency virus type 1 integrase D,D(35)E motif do not eliminate provirus formation

The core domain of human immunodeficiency virus type 1 (HIV-1) integrase (IN) contains a D,D(35)E motif, named for the phylogenetically conserved glutamic acid and aspartic acid residues and the invariant 35 amino acid spacing between the second and third acidic residues. Each acidic residue of the D,D(35)E motif is independently essential for the 3'-processing and strand transfer activities of purified HIV-1 IN protein. Using a replication-defective viral genome with a hygromycin selectable marker, we recently reported that a mutation at any of the three residues of the D,D(35)E motif produces a 10(3)- to 10(4)-fold reduction in infectious titer compared with virus encoding wild-type IN (A. D. Leavitt et al., J. Virol. 70:721-728. 1996). The infectious titer, as measured by the number of hygromycin-resistant colonies formed following infection of cells in culture, was less than a few hundred colonies per microg of p24. To understand the mechanism by which the mutant virions conferred hygromycin resistance, we characterized the integrated viral DNA in cells infected with virus encoding mutations at each of the three residues of the D,D(35)E motif. We found the integrated viral DNA to be colinear with the incoming viral genome. DNA sequencing of the junctions between integrated viral DNA and host DNA showed that (i) the characteristic 5-bp direct repeat of host DNA flanking the HIV-1 provirus was not maintained, (ii) integration often produced a deletion of host DNA, (iii) integration sometimes occurred without the viral DNA first undergoing 3'-processing, (iv) integration sites showed a strong bias for a G residue immediately adjacent to the conserved viral CA dinucleotide, and (v) mutations at each of the residues of the D,D(35)E motif produced essentially identical phenotypes. We conclude that mutations at any of the three acidic residues of the conserved D,D(35)E motif so severely impair IN activity that most, if not all, integration events by virus encoding such mutations are not IN mediated. IN-independent provirus formation may have implications for anti-IN therapeutic agents that target the IN active site.     

Autonomic components of the cardiovascular responses to an acoustic startle stimulus in rats

The Afa-family repetitive sequences were isolated from barley (Hordeum vulgare, 2n = 14) and cloned as pHvA14. This sequence distinguished each barely chromosome by in situ hybridization. Double color fluorescence in situ hybridization using pHvA14 and 5S rDNA or HvRT-family sequence (subtelomeric sequence of barley) allocated individual barley chromosomes showing a specific pattern of pHvA14 to chromosome 1H to 7H. As the case of the D genome chromosomes of Aegilops squarrosa and common wheat (Triticum aestivum) hybridized by its Afa-family sequences, the signals of pHvA14 in barley chromosomes tended to appear in the distal regions that do not carry many chromosome band markers. In the telomeric regions these signals always placed in more proximal portions than those of HvRT-family. Based on the distribution patterns of Afa-family sequences in the chromosomes of barley and D genome chromosomes of wheat, we discuss a possible mechanism of amplification of the repetitive sequences during the evolution of Triticeae. In addition, we show here that HvRT-family also could be used to distinguish individual barley chromosomes from the patterns of in situ hybridization.     

Functional interactions of the HHCC domain of moloney murine leukemia virus integrase revealed by nonoverlapping complementation and zinc-dependent dimerization

The retroviral integrase (IN) is required for the integration of viral DNA into the host genome. The N terminus of IN contains an HHCC zinc finger-like motif, which is conserved among all retroviruses. To study the function of the HHCC domain of Moloney murine leukemia virus IN, the first N-terminal 105 residues were expressed independently. This HHCC domain protein is found to complement a completely nonoverlapping construct lacking the HHCC domain for strand transfer, 3' processing and coordinated disintegration reactions, revealing trans interactions among IN domains. The HHCC domain protein binds zinc at a 1:1 ratio and changes its conformation upon binding to zinc. The presence of zinc within the HHCC domain stimulates selective integration processes. Zinc promotes the dimerization of the HHCC domain and protects it from N-ethylmaleimide modification. These studies dissect and define the requirement for the HHCC domain, the exact function of which remains unknown.     

Redesigning primary care processes to improve the offering of mammography. The use of clinic protocols by nonphysicians

We have isolated a genomic clone corresponding to a caffeic acid O-methyltransferase (COMT) from barley (Hordeum vulgare L.) using a cDNA for a previously described jasmonate-regulated mRNA showing homology to COMT. Primer extension was used to characterize the 5' end of the mRNA while the 3' end, intron/exon structure and other features of the sequence were deduced by comparison to the cDNA sequence and/or conserved motifs. The gene is mapped to chromosome five and is absent in the barley cultivar Morex. Southern and northern analyses suggest that no differences in genomic structure and jasmonate inducibility exist between the barley cultivar Salome (source of the cDNA clone) and Igri (source of the genomic clone). This genomic clone is thus suitable for promoter studies with respect to jasmonate induction.     

Dicaffeoylquinic acid inhibitors of human immunodeficiency virus integrase: inhibition of the core catalytic domain of human immunodeficiency virus integrase

Integration of a cDNA copy of the human immunodeficiency virus (HIV) genome is mediated by an HIV-1-encoded enzyme, integrase (IN), and is required for productive infection of CD4+ lymphocytes. It had been shown that 3,5-dicaffeoylquinic acid and two analogues were potent and selective inhibitors of HIV-1 IN in vitro. To determine whether the inhibition of IN by dicaffeoylquinic acids was limited to the 3,5 substitution, 3,4-, 4,5-, and 1,5-dicaffeoylquinic acids were tested for inhibition of HIV-1 replication in tissue culture and inhibition of HIV-1 IN in vitro. All of the dicaffeoylquinic acids were found to inhibit HIV-1 replication at concentrations ranging from 1 to 6 microM in T cell lines, whereas their toxic concentrations in the same cell lines were > 120 microM. In addition, the compounds inhibited HIV-1 IN in vitro at submicromolar concentrations. Molecular modeling of these ligands with the core catalytic domain of IN indicated an energetically favorable reaction, with the most potent inhibitors filling a groove within the predicted catalytic site of IN. The calculated change in internal free energy of the ligand/IN complex correlated with the ability of the compounds to inhibit HIV-1 IN in vitro. These results indicate that the dicaffeoylquinic acids as a class are potent and selective inhibitors of HIV-1 IN and form important lead compounds for HIV drug discovery.