Replication,maturation and physical mapping of bacteriophage MB78 genome

Bacteriophage MB78 is a virulent phage ofSalmonella typhimurium. The viral DNA is 42 kb in size and seems to be circularly permuted. We show that viral DNA replication is through concatemeric DNA formation which is subsequently converted into full length DNA through headful packaging. A restriction map of MB78 DNA for six restriction endonucleases e.g.BgIII,PvuII, ECORI, ClaI, SalI and SmaI has been constructed. The yield of certain fragments in less than molar amount is explained in terms of permutation and the headful mechanism of packaging. The packaging site (pac site) has been suggested.     

Termini and telomeres in T-DNA transformation

A T-DNA vector for plant transformation has been constructed in which the cloning site is located 9 bp from the right-border (RB) end and 27 bp from the left-border (LB) end. In this vector cloned DNA homologous to plant chromosomal sequences is located at the T-DNA termini, and will thus be exposed by even limited exonucleolysis in planta. The arabidopsis ADH (alcohol dehydrogenase) locus was mobilized from Agrobacterium, and integration into the recipient genome was studied. Despite the terminal location of ADH homology in this vector, the T-DNA integrated essentially at random in the Arabidopsis genome rather than at the endogenous ADH locus. T-DNA integration was blocked, however, when Arabidopsis telomeric sequences were added to the construct at each end of the ADH homology. Thus the predominant mode by which incoming T-DNA is integrated into the continuity of chromosomal DNA involves free DNA ends, but, in contrast to modes of recombination such as gap repair, does not involve extensive terminal DNA sequence homology.     

Virus DNA packaging: the strategy used by phage λ

Phage λ, like a number of other large DNA bacterio-phages and the herpesviruses, produces concatemeric DNA during DNA replication. The concatemeric DNA is processed to produce unit-length, virion DNA by cutting at specific sites along the concatemer. DNA cutting is coordinated with DNA packaging, the process of translocation of the cut DNA into the preformed capsid precursor, the prohead. A key player in the λ DNA packaging process is the phage-encoded enzyme terminase, which is involved in (i) recognition of the concatemeric λ DNA; (ii) initiation of packaging, which includes the introduction of staggered nicks at cosN to generate the cohesive ends of virion DNA and the binding of the prohead; (iii) DNA packaging, possibly including the ATP-driven DNA translocation; and (iv) following translocation, the cutting of the terminal cosN lo complete DNA packaging. To one side of cosN is the site cosB, which plays a role in the initiation of packaging; along with ATP, cosB stimulates the efficiency and adds fidelity to the endo-nuclease activity of terminase in cutting cosN. cosB is essential for the formation of a post-cleavage complex with terminase, complex I, that binds the prohead, forming a ternary assembly, complex II. Terminase interacts with cosN through its large subunit, gpA, and the small terminase subunit, gpNul, interacts with cosB. Packaging follows complex II formation. cosN is flanked on the other side by the site cosQ, which is needed for termination, but not initiation, of DNA packaging. cosQ is required for cutting of the second cosN, i.e. the cosN at which termination occurs. DNA packaging in λ has aspects that differ from other λ DNA transactions. Unlike the site-specific recombination system of λ, for DNA packaging the initial site-specific protein assemblage gives way to a mobile, translocating complete, and unlike the DNA replication system of λ, the same protein machinery is used for both initiation and translocation during λ DNA packaging.     

The cis-acting DNA sequences required in vivo for bacteriophage Mu helper-mediated transposition and packaging

The 37,000 bp double-stranded DNA genome of bacteriophage Mu behaves as a plaque-forming transposable element of Escherichia coli. We have defined the cis-acting DNA sequences required in vivo for transposition and packaging of the viral genome by monitoring the transposition and maturation of Mu DNA-containing pSC101 and pBR322 plasmids with an induced helper Mu prophage to provide the trans-acting functions. We found that nucleotides 1 to 54 of the Mu left end define an essential domain for transposition, and that sequences between nucleotides 126 and 203, and between 203 and 1,699, define two auxiliary domains that stimulate transposition in vivo. At the right extremity, the essential sequences for transposition require not more than the first 62 base pairs (bp), although the presence of sequences between 63 and 117 bp from the right end increases the transposition frequency about 15-fold in our system. Finally, we have delineated the pac recognition site for DNA maturation to nucleotides 32 to 54 of the Mu left end which reside inside of the first transposase binding site (L1) located between nucleotides 1–30. Thus, the transposase binding site and packaging domains of bacteriophage Mu DNA can be separated into two well-defined regions which do not appear to overlap.Abbreviations attL attachment site left - attR attachment site right - bp base pairs - Kb kilobase pair - nt nucleotide - Pu Purine - Py pyrimidine - Tn transposable element State University of New York, Downstate Medical Center, Brooklyn, NY 11204 USA     

Physical mapping of the BglI, BglII, PstI and EcoRI restriction fragments of staphylococcal phage phi 11 DNA

Summary A cleavage map of the generalized transducing staphylococcal phage 11 DNA has been constructed by reciprocal double digestion. All three BglI, the six BglII, the three PstI, and 11 out of 15 EcoRI sites have been mapped. The map is circular, with a total length of 42 kb, and has been divided into 100 map units. The phage DNA is cyclically permuted and has a terminal redundancy of about 11 kb. The preferential starting point and direction for packaging DNA lies at map unit 79 and proceeds towards higher map units.     

Xenocylindrocladium guianense andX. subverticillatum, two new species of hyphomycetes from plant debris in the tropics

Two new species of hyphomycetes,Xenocylindrocladium guianense andX. subverticillatum, are described from plant debris collected in French Guiana and Singapore, respectively. The genusXenocylindrocladium has thus far been known from one species,X. serpens, which was described from plant debris collected in Ecuador. The two new taxa are compared with and distinguished fromX. serpens based on morphology, cultural characteristics and phylogenetic analysis of DNA sequence data of the 5.8S rDNA with flanking ITS1 and ITS2 regions and the 5′ end of the β-tubulin gene. These species are also compared with other closely related hypocrealean taxa. Present collection data suggest that species ofXenocylindrocladium could be restricted to the tropics.     

Organization and chromosomal localization ofβ-tubulin genes inLeishmania donovani

The genomic organization and chromosomal location of theβ-tubulin isogenes inLeishmania donovani promastigotes has been studied by nucleic acid hybridization techniques using a cloned β-tubulin gene. We have cloned aβ-tubulin gene fragment, 3.3 kbp long, from genomic DNA ofLeishmania donovani using a heterologousβ-tubulin DNA as probe. Restriction maps of this clone have been prepared. It has been estimated that there are approximately 11–15 copies of theβ-tubulin genes per haploid genome. The majority of these isogenes are arranged in a tandem repeat with a length of 3.5 kbp on a single chromosome. In addition a few dispersed gene copies at different chromosomal loci were detected by pulse field gradient gel electrophoresis. Part of the internal coding region of the gene has been sequenced to confirm the identity of theβ-tubulin clone and is found to be nearly identical to that ofLeishmania mexicana amazonensis.     

Functional characterization of a chicken major histocompatibility complex class II B gene promoter

 A 0.7 kilobase (kb) DNA fragment from the 5′ flanking region of a chicken major histocompatibility complex (MHC) class II B gene was cloned into chloramphenicol acetyltransferase (CAT) reporter vectors and was transfected into a chicken macrophage cell line that expresses a low level of MHC class II antigens. Positive orientation-dependent promoter activity of the chicken DNA was evident in a reporter construct containing an SV40 enhancer. Deletion analysis of this 0.7 kb DNA fragment revealed a short fragment in the 3′ end that was crucial for the promoter function and negative regulatory elements (NRE) located further upstream. The conserved MHC class II X and Y boxes did not have a significant effect on promoter activity. Sequence analysis of the 0.7 kb class II B gene upstream region suggests possible involvement of interferon (IFN), E twenty-six specific (ETS)-related proteins, and other factors in regulating this promoter. A chicken T-cell line culture supernatant increased surface expression of MHC class II antigens, as well as class II promoter activity, in this macrophage cell line. This first functional characterization of a chicken MHC class II B gene promoter will aid in understanding the regulatory mechanisms that control the expression of these genes. Received: 9 July 1996 / Revised: 7 October 1996     

Biochemical,genetic and molecular characterization of new respiratory-deficient mutants inChlamydomonas reinhardtii

Eight respiratory-deficient mutants ofChlamydomonas reinhardtii have been isolated after mutagenic treatment with acriflavine or ethidium bromide. They are characterized by their inability to grow or their very reduced growth under heterotrophic conditions. One mutation (Class III) is of nuclear origin whereas the seven remaining mutants (Classes I and II) display a predominantly paternalmt ^- inheritance, typical of mutations residing in the mitochondrial DNA. Biochemical analysis has shown that all mutants are deficient in the cyanide-sensitive cytochrome pathway of the respiration whereas the alternative pathway is still functional. Measurements of complexes II + III (antimycin-sensitive succinate-cytochromec oxido-reductase) and complex IV (cytochromec oxidase) activities allowed to conclude that six mutations have to be localized in the mitochondrial apocytochromeb (COB) gene, one in the mitochondrial cytochrome oxidase subunit I (COI) gene and one in a nuclear gene encoding a component of the cytochrome oxidase complex. By using specific probes, we have moreover demonstrated that five mutants (Class II mutants) contain mitochondrial DNA molecules deleted in the terminal end containing the COB gene and the telomeric region; they also possess dimeric molecules resulting from end-to-end junctions of deleted monomers. The two other mitochondrial mutants (Class I) have no detectable gross alteration. Class I and Class II mutants can also be distinguished by the pattern of transmission of the mutation in crosses.Anin vivo staining test has been developed to identify rapidly the mutants impaired in cyanide-sensitive respiration.     

Bioreduction of Cu(II) by Cell-Free Copper Reductase from a Copper Resistant <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. NA

Environmental copper contamination is a serious human health problem. Copper reductase is produced by microorganisms to facilitate copper uptake by ATPases into the cells increasing copper biosorption. This study assessed the reduction of Cu(II) by cell-free extracts of a highly copper-resistant bacterium, Pseudomonas sp. strain NA, isolated from vineyard soil contaminated with copper. Both intact cells and cell-free extract of Pseudomonas sp. strain NA displayed substantial reduction of Cu(II). Intact cells reduced more then 80 mg L⁻¹ of Cu(II) from medium amended with 200 mg L⁻¹ of copper after 24 h of incubation. Cell-free extract of the isolate reduced more than 65% of the Cu(II) at initial copper concentration of 200 mg L⁻¹ after 24 h. Soluble protein production was high at 72 h of incubation at 100 mg L⁻¹ of copper, with more then 60 μg L⁻¹ of total soluble protein in cell-free extract recorded. Cu(II) reduction by isolate NA was increased when copper concentration increased for both intact cells and cell-free extract. Results indicate that Pseudomonas sp. strain NA produces copper reductase enzyme as the key mechanism of copper biotransformation.     

N-[2-Naphthyl]-glycine hydrazide,a potent inhibitor of DNA-dependent RNA polymerase ofMycobacterium tuberculosis H37RV

N-[2-Naphthyl]-glycine hydrazide has been shown for the first time as a potent inhibitor of the DNA-dependent RNA polymerase (EC 2.7.7.6) ofMycobacterium tuberculosis H₃₇R_v. At a concentration of 10^-9 M, the compound shows maximum inhibition of the enzyme, the inhibition being less at higher concentrations. It is suggested that the novel type of inhibition pattern may be due to hydrophobic interactions occurring between the molecules of the compound at higher concentrations. The finding that there is a shift in the λ_max of the compound could also account for this phenomenon. The effect of this compound was also tested on DNA-dependent RNA polymerases from an eukaryotic fungus,Microsporum canis. At a concentration of 10⁻⁹ M it inhibits RNA polymerase II (32%) but not RNA polymerasesI andIII     

Karyotyping of Brassica oleracea L. based on rDNA and Cot -1 DNA fluorescence in situ hybridization

To explore an effective and reliable karyotyping method in Brassica crop plants, Cot-1 DNA was isolated from Brassica oleracea genome, labeled as probe with Biotin-Nick Translation Mix kit, in situ hybridized to mitotic spreads, and where specific fluorescent bands showed on each chromosome pair. 25S and 5S rDNA were labeled as probes with DIG-Nick Translation Mix kit and Biotin-Nick Translation Mix kit, respectively, in situ hybridized to mitotic preparations, where 25S rDNA could be detected on two chromosome pairs and 5S rDNA on only one. Cot-1 DNA contains rDNA and chromosome sites identity between Cot-1 DNA and 25S rDNA was determined by dual-colour fluorescence in situ hybridization. All these showed that the karyotyping technique based on a combination of rDNA and Cot-1 DNA chromosome landmarks is superior to all but one. A more exact karyotype of B. oleracea has been analyzed based on a combination of rDNA sites, Cot-1 DNA fluorescent bands, chromosome lengths and arm ratios. __________ Translated from Journal of Wuhan University (Nat. Sci. Ed.), 2006, 52(2): 230–234 [译自: 武汉大学学报 (理学版)]     

Characterization and chromosome location of satellite DNA in the leaf beetle Chrysolina americana (Coleoptera, Chrysomelidae)

This paper is the first record of the satellite DNA of the specialized phytophagous genus Chrysolina. The satellite DNA of Chrysolina americana is organized in a tandem repeat of monomers 189 bp long, has a A + T content of 59.6 % and presents direct and inverted internal repeats. Restriction analysis of the total DNA with methylation sensitive enzymes suggests that this repetitive DNA is undermethylated. In situ hybridization with a biotinylated probe of the satellite DNA showed the pericentromeric localization of these sequences in all meiotic bivalents. The presence of this repetitive DNA in other species of the genus was also tested by Southern analysis. The results showed that this satellite DNA sequence is specific to the C. americana genome and has not been found in three other species of Chrysolina with a different choice of host plants than in the former. This revised version was published online in July 2006 with corrections to the Cover Date.     

Heterotrophic nitrogen fixation in oligotrophic tropical marshes: changes after phosphorus addition

In order to determine the impact of nutrient enrichment on phosphorus (P) limited wetlands, we established experimental P additions in marshes throughout northern Belize. P significantly increased macrophyte primary production, which led to the rapid elimination of cyanobacterial mats. The replacement of cyanobacterial mats by macrophytes constrained autotrophic nitrogen (N) fixation, increased the quantity, and changed the quality of organic matter input to the sediments. We predicted that the activity of sediment heterotrophic N fixers will be impacted by these alterations in carbon input. We used the acetylene reduction technique to measure potential (glucose amended) nitrogenase activity (NA) in sediments from controls and treatment plots that have been P enriched for four years and dominated either by Eleocharis cellulosa, or Typha domingensis for two years. NA in P-enriched plots was 2–3 orders of magnitude higher than NA in controls. NA was positively correlated with the soil reactive P, both total organic and microbial carbon, live root biomass, and total phospholipid fatty acids (PLFA) as an indicator of active microbial biomass. It was negatively correlated with the concentration of ammonium-N. Path analysis revealed that the indirect effect of P on NA through the root biomass was more important than the direct effect of P. NA of the upper sediment layer was consistently higher in Eleocharis than in Typha dominated plots, despite the higher litter input by Typha. We feel that the higher levels of lignin and phenolics occurring in Typha litter, relative to Eleocharis, constrained NA in Typha plots. Handling editor: Luis Mauricio Bini     

Biochemical evidence of a physical interaction between <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis> B-family and Y-family DNA polymerases

The hyper-thermophilic archaeon Sulfolobus solfataricus possesses two functional DNA polymerases belonging to the B-family (Sso DNA pol B1) and to the Y-family (Sso DNA pol Y1). Sso DNA pol B1 recognizes the presence of uracil and hypoxanthine in the template strand and stalls synthesis 3–4 bases upstream of this lesion (“read-ahead” function). On the other hand, Sso DNA pol Y1 is able to synthesize across these and other lesions on the template strand. Herein we report evidence that Sso DNA pol B1 physically interacts with DNA pol Y1 by surface plasmon resonance measurements and immuno-precipitation experiments. The region of DNA pol B1 responsible for this interaction has been mapped in the central portion of the polypeptide chain (from the amino acid residue 482 to 617), which includes an extended protease hyper-sensitive linker between the N- and C-terminal modules (amino acid residues Asn482-Ala497) and the α-helices forming the “fingers” sub-domain (α-helices R, R′ and S). These results have important implications for understanding the polymerase-switching mechanism on the damaged template strand during genome replication in S. solfataricus.     

In vivo loss of supercoiled DNA carrying a palindromic sequence

Summary Interest in the fate of long palindromic DNA sequences in E. coli has been kindled by the observation that their inviability is overcome in recBC sbcB strains and that these hosts permit the construction of DNA libraries containing long palindromic sequences present in the human genome. In this paper we show that a reduction in the level of intracellular supercoiled DNA occurs as the result of the presence of a 530 bp palindrome in bacteriophage lambda. This reduction occurs in Rec⁺ and recA strains but not in strains lacking exonucleases V and I (recBC sbcB). However, the DNA must be active (not repressed) for this reduction to be observed, since it is not seen in a Rec⁺ host lysogenic for phage lambda. These results argue against two hypotheses: firstly, that the palindrome causes inviability solely by interfering with packaging, and secondly, that it dose so solely by interfering with recombination. Conversely, these results suggest that a feature of active monomeric DNA (probably its replication) is involved in inviability.     

巴西橡胶树栽培种质基因组C值测定和变异分析

为了解巴西橡胶树(Hevea brasiliensis)栽培种质的变异情况,以53份在云南植胶区综合性状表现较好的巴西橡胶树栽培种质为材料,采用流式细胞术测定了基因组C值,并进行了变异分析。结果表明,浅绿色嫩叶是巴西橡胶树流式细胞术测定的最适样品。53份巴西橡胶树栽培种质的细胞核DNA含量和基因组C值存在一定差异,基因组的平均C值是1.531 696×109 bp,最小的是CRTG-272种质(1.465 908×10~9 bp),最大的是CRTG-83种质(1.600 381×10~9 bp),变异系数较小(CV=0.035 5)。53份巴西橡胶树栽培种质中有47份为二倍体,6份为三倍体。在已测定基因组大小的40种大戟科(Euphorbiaceae)植物中,基因组大小变异较大(CV=1.248 6),与"C值悖论"观点相一致。因此,应用流式细胞术能快速、准确地测定巴西橡胶树细胞核DNA含量、基因组C值和染色体倍性。     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Hyacinthus orientalis</Emphasis> with thaumatin II gene to control fungal diseases

Transgenic plants of hyacinth (Hyacinthus orientalis L.) cvs. Edisson and Chine Pink have been obtained by Agrobacterium-mediated transformation. Leaf explants of the both hyacinth cultivars regenerated shoots on MS medium containing 2.2 μM BAP and 0.3 μM NAA at a frequency of 95%. A. tumefaciens strain CBE21 carrying binary vector pBIThau35 was used for transformation. Plasmid pBIThau35 has been produced by cloning preprothaumatin II cDNA into pBI121 instead of uidA gene. Inoculated leaf explants formed calli and shoots at high frequency on selective medium with 100 mg l⁻¹ kanamycin. Four hyacinth transgenic lines of cv. Chine Pink and one line of cv. Edisson have been selected on medium containing 200 mg l⁻¹ kanamycin. The insertion of thaumatin II gene into hyacinth genome has been confirmed by PCR-analysis. All transgenic plants expressed substantial amounts of thaumatin II (between 0.06 and 0.28% of the total soluble protein). Hyacinth transgenic lines were assayed for resistance to the pathogenic fungi Fusarium culmorum and Botrytis cinerea. There were no significant differences between nontransformed control and transgenic leaves of both cultivars. At the same time the bulbs of the transgenic line Н7401 cv. Chine Pink showed the higher level of resistance to B. cinerea, the bulbs of the transgenic line Н7404 were more resistant to F. culmorum. In both cases the signs of the fungal disease were developed more slowly. The resistance of the bulbs cv. Edisson line to these fungi was not changed. All transgenic hyacinth plant were successfully transferred to soil for further evaluation.