Antagonistic properties of two recombinant strains of Streptomyces melanosporofaciens obtained by intraspecific protoplast fusion.

Intraspecific protoplast fusion was used to produce stable prototrophic recombinants of Streptomyces melanosporofaciens EF-76, a biocontrol agent of plant disease producing geldanamycin. Two recombinant strains (FP-54 and FP-60) that differed with regard to their antagonistic properties against Bacillus cereus ATCC 14579, Streptomyces scabies EF-35 and Phytophthora fragariae var. rubi 390 were characterized. FP-60 lost the ability to inhibit the in vitro growth of these microbial strains while FP-54 exhibited higher antagonistic activities against them. FP-60 was deficient in geldanamycin biosynthesis whereas FP-54 was shown to produce, in addition to geldanamycin, at least two other antimicrobial compounds that were absent in the culture supernatants of strain EF-76. Like the wild-type strain EF-76, strain FP-54 reduced common scab symptoms on potato tuber but no significant difference was observed between the disease index attributed to tubers treated with strain EF-76 or with strain FP-54. Strain FP-60 showed no protective effect against common scab. The disease index of tubers treated with this recombinant was worse than the index associated with potato tubers from control treatments.

Characterization of bifidobacteria by random DNA amplification.

RAPD conditions were optimized to generate reproducible banding patterns by testing primers, thermocyclers and overall reproducibility in repeat DNA analysis and separate DNA extractions. Five primers were chosen on the basis of band intensity and distribution (between 2 and 10 bands) which clearly distinguished among strains of Bifidobacterium adolescentis, B. animalis, B. bifidum, B. breve, B. infantis and B. longum. The use of five single-primer reactions under optimized conditions improved the resolution and accuracy of the RAPD method for the characterization of dairy-related bifidobacteria. The results indicated that this method was highly reproducible in repeated analysis. Similarity between bifidobacteria strains was evaluated based on their RAPD profiles. Using a set of five primers, it was demonstrated that it may be possible to distinguish three different species of Bifidobacterium (B. breve, B. bifidum and B. adolescentis), based on similarity of the RAPD profiles to known reference strains. Furthermore, application of the RAPD technique may also be useful and faster, than traditional systematics for placement of industrial strains into specific clusters (either B. longum/infantis or B. animalis/lactis).

Glucanolytic Actinomycetes Antagonistic to Phytophthora fragariae var. rubi, the Causal Agent of Raspberry Root Rot.

A collection of about 200 actinomycete strains was screened for the ability to grow on fragmented Phytophthora mycelium and to produce metabolites that inhibit Phytophthora growth. Thirteen strains were selected, and all produced (beta)-1,3-, (beta)-1,4-, and (beta)-1,6-glucanases. These enzymes could hydrolyze glucans from Phytophthora cell walls and cause lysis of Phytophthora cells. These enzymes also degraded other glucan substrates, such as cellulose, laminarin, pustulan, and yeast cell walls. Eleven strains significantly reduced the root rot index when inoculated on raspberry plantlets.

Saccharopolyspora hirsuta 367 encodes clustered genes similar to ketoacyl synthase, ketoacyl reductase, acyl carrier protein, and biotin carboxyl carrier protein.

The actI gene, encoding a component of the actinorhodin polyketide synthase of Streptomyces coelicolor, was used to identify and clone a homologous 11.7 kb BamHI DNA fragment from Saccharopolyspora hirsuta 367. The cloned fragment complemented actinorhodin production in a strain of Streptomyces coelicolor bearing a mutant actI gene. The DNA sequence of a 5.1 kb fragment revealed 6 open reading frames (ORF). ORF1 does not resemble any known DNA or deduced protein sequence, while the deduced protein sequence of ORF2 resembles that of biotin carboxyl carrier proteins. Based on the similarity to deduced protein sequences from cloned genes of polyketide producers, ORF3 would code for a ketoreductase, ORF4 and ORF5 for the putative heterodimeric beta-ketoacyl synthase, and ORF6 for an acyl carrier protein.

A cloned replicon of Saccharopolyspora phages JHJ-1 and JHJ-3 is stably maintained as a plasmid in various actinomycetes.

A replicon of phage JHJ-1 (and JHJ-3) was cloned. The autonomously replicating phage element was maintained as a medium-copy-number shuttle plasmid in many actinomycetes, and was efficiently transmitted to spores without antibiotic selection. One gene was shown to be expressed in a vector containing the JHJ-3 replicon.

Construction of a shuttle lacZ alpha-based Escherichia coli-actinomycetes vector containing the phage JHJ-3 replicon.

Using the broad replicating range JHJ-3 phage replicon, a shuttle vector for Escherichia coli and actinomycetes has been constructed. The vector, pOJ31, bears the lacZ alpha fragment allowing a blue/white gene cloning system. pOJ31 also contains a polylinker of 15 unique cloning sites and the phage T7 promoter. The vector has been used to stably express the mel gene from plasmid pIJ702 in Streptomyces lividans.

Biological characterization of induced phages from Saccharopolyspora hirsuta 367 and comparison with phage JHJ-1.

Phages JHJ-2 and JHJ-3 were isolated from Saccharopolyspora hirsuta 367 UC 8106 following induction with mitomycin C and amplified on S. hirsuta NRRL B-5792. Their properties were compared with those of phage JHJ-1, isolated previously from S. hirsuta 367 NRRL 12045. The DNA restriction patterns appeared to be identical. One-step growth experiments showed no differences between the replication cycles. Burst sizes ranged from 100 to 110 p.f.u. per cell. However, the three phages showed some differences in their behaviour in different hosts. The host range of phage JHJ-1, on non-lysogenic strains, was emended to include all of the Saccharopolyspora strains tested; the host range of phage JHJ-2 was shown to be identical to JHJ-1. Phage JHJ-3 did not form detectable plaques on strains of S. rectivirgula or S. erythraea except S. erythraea NRRL 2359. Neither phage JHJ-2 nor JHJ-3 formed plaques on any lysogenic strains, while JHJ-1 formed plaques on all such strains except S. hirsuta 367 UC8106. Phage JHJ-3 was characterized as a temperate bacteriophage because it formed turbid, self-limiting plaques and lysogenized S. hirsuta NRRL B-5792. It was spontaneously released from UC8106. Both JHJ-1 and JHJ-2 formed clear and invasive (Inv+ phenotype: the property to grow on old mycelium) plaques on some Saccharopolyspora strains but clear and self-limiting plaques on others. Thus, the expression of the Inv+ phenotype encoded by JHJ-1 and JHJ-2 appears to be modulated by the host cell.

Doppler umbilical artery velocimetry in fetuses with polyhydramnios.

Polyhydramnios is a condition of multiple etiologies, many of a benign nature, but some of which are incompatible with life. To evaluate Doppler velocimetry results as a prognostic parameter in these fetuses, we reviewed all of our cases of polyhydramnios that underwent Doppler analysis in the third trimester. Fifty-four fetuses were studied. Eleven (20.4%) had abnormal waveforms and 43 (79.6%) had normal waveforms. An abnormal waveform was associated with a significantly higher incidence of congenital anomalies, perinatal mortality and intrauterine growth retardation. Six of the 11 fetuses had abnormal karyotypes. Macrosomia was present in 37.2% of fetuses with normal waveforms and in no fetus with an abnormal waveform. Doppler analysis may aid in the counseling and management of patients with polyhydramnios. In cases with an abnormal ratio, the physician and patients should be prepared for a poor outcome and third trimester genetic analysis should be strongly considered.

Amniotic fluid index: an appropriate predictor of perinatal outcome.

Qualitative amniotic fluid volume determination is a routine part of the fetal biophysical profile score. Quantitative amniotic fluid volume measurement, however, is not a factor in the determination of the standard biophysical profile score. This study is a retrospective analysis of antepartum assessment of amniotic fluid volumes and their relationship to neonatal outcomes. The amniotic fluid index was calculated for all patients examined and perinatal outcome was studied for all patients assessed. Patients with reduced or increased amniotic fluid volume had a significant increase in meconium-stained amniotic fluid, Apgar scores less than 7 at 1 and 5 minutes, major congenital anomalies, admission to the neonatal intensive care unit, and were more likely to require delivery by cesarean section for fetal distress. This study suggests that a quantitative ultrasound measurement of amniotic fluid volume represents an effective discriminatory test to be used in pregnancy evaluation.

Saccharopolyspora hirsuta strain 367 releases JHJ-1, a bacteriophage capable of propagation on old mycelium.

Bacteriophage JHJ-1 was isolated from Saccharopolyspora hirsuta strain 367 NRRL 12045 as an endogenous but virulent phage. The plaque size was not self-limiting, since a few p.f.u. could completely lyse a lawn. Electron microscopy showed that this phage belonged to group B of Bradley's morphological classification. The JHJ-1 genome is a linear DNA molecule of 41.1 kbp with cohesive ends and a G + C content of 68.8-70.0 mol%. The DNA cleavage map was established for 12 restriction endonucleases. The host range is apparently very narrow, being limited to two strains of S. hirsuta (NRRL 12045 and NRRL B-5792). However, JHJ-1 did not lytically infect S. hirsuta strain 367 UC 8106. Phage JHJ-1 was shown, by Southern blot analysis, to lysogenize both S. hirsuta NRRL 12045 and UC 8106. It thus appears to behave as a virulent mutant of a temperate phage on one, but not on the other, JHJ-1 lysogen.

Characterization of SE-3, a virulent bacteriophage of Saccharopolyspora erythraea.

SE-3 is a virulent bacteriophage isolated from a large-scale culture of Saccharopolyspora erythraea, an erythromycin producer. The host range of the phage is narrow, limited to some strains of this species. Another strain of Sac. erythraea, and a strain of Sac. hirsuta, are able to adsorb phage particles but do not sustain their complete multiplication. SE-3 is closely related to the phage SE-5 as shown by DNA restriction mapping. The differences between SE-3 and SE-5 genomes are apparently limited to two DNA segments flanked by short inverted repeats, visualized by electron microscopy.

Activation of the major late promoter in adenovirus transformed cells by 5-azacytidine.

The adenovirus major late promoter (MLP) is normally not active in transformed cells. We investigated if it could be activated with 5-azacytidine. Three days of treatment with 10 microM 5-azacytidine induced transient activation of the MLP as shown by hybridization with an L1 r-strand-specific probe. The po/III-transcribed VA-RNAs were not activated. L1 activation was not accompanied by detectable changes in methylation of HpaII sites at the promoter or in the body of the transcript. Stably activated cell clones could be obtained at 20% frequency after long-term drug treatment.

Agranulocytosis associated with sulfasalazine.

Agranulocytosis is a rare but potentially lethal adverse effect of sulfasalazine. We report a case of sulfasalazine-associated agranulocytosis that occurred in a 79-year-old woman who had been taking the drug for approximately seven weeks. The patient had discontinued the drug on her own initiative nine days prior to admission. The patient was admitted with complaints of hoarseness, fever, odynophagia, and malaise. The total white blood cell count was 600/mm3 with a differential of 0% neutrophils, 8% bands, 67% lymphocytes, and 25% monocytes; a bone marrow aspirate and biopsy revealed maturation arrest. The patient's peripheral white blood cell count and differential progressively increased over the nine-day hospital course. Upon discharge the white blood cell count was 12,000 cells/mm3 with 66% neutrophils, 8% bands, 16% lymphocytes, and 10% monocytes. Complete blood counts should be performed periodically in patients receiving sulfasalazine, especially during the first two months of therapy. Pharmacists should counsel patients to discontinue the drug and consult their physician immediately if they develop unexplained fever, chills, sore throat, malaise, or other nonspecific illness during the initial two months of treatment.

Response of individual adenovirus promoters to the products of the E1A gene.

Adenovirus E1A genes possess transcriptional activation and repression activities. Three major gene products have been characterized, derived from the 13S, 12S and 9S mRNAs. Using transient expression assays in HeLa cells, we have investigated the effect of these gene products on the activity of the nine major Ad2 (or Ad5) promoters driving the expression of a reporter gene. Based on the results, we could separate the promoters into three classes: (a) E1A, which is active by itself, and is unaffected or slightly stimulated by the E1A 13S product (depending on the HeLa cell line used); (b) the other classical early promoters (E1B, E2e, E3, E4), all of which are active alone and are stimulated by the 13S product and repressed by the 12S product; and (c) the late promoters (IX, IVa2, MLP, E2L) which are not active alone and are substantially unaffected by the 13S or 12S products. Thus the 13S and 12S gene products have antagonistic effects on at least four adenovirus promoters. The 9S product did not influence the activity of any of the adenovirus promoters. Upon transfection into 293 cells, all the early promoters were active and all the late promoters were inactive, except for the major late promoter (MLP). We demonstrate that the combination of the E1A and E1B genes is a potent activator of the MLP in HeLa cells and discuss these results in the context of the infectious cycle.

Transactivation of host and viral genes by the adenovirus E1B 19K tumor antigen.

Adenovirus contains two nuclear oncogenes, the EIA and EIB genes, which cooperatively can transform cells through mechanisms that are not understood. The transcriptional activities of the E1A gene (transactivation and repression) are well studied. Using transient expression assays, we show here that the 19,000-Da E1B gene product can also activate all the adenovirus early promoters (E1A, E1B, E2e, E3 and E4) and a cellular heat shock gene promoter (hsp70), but not the adenovirus late promoters (IX, IVa2, MLP and E2L). The effect is greatest under conditions where cell growth is inhibited, and appears to operate at the transcriptional level. Possible interactions with enhancer elements are discussed. Although the E1B stimulatory effect does not require the presence of E1A gene products, a synergistic effect is obtained in the presence of E1A 13S product. This activity of the E1B gene is also observed during virus infection and is likely to have important consequences in lytically infected and transformed cells.

Possible role of ADP-ribosylation of adenovirus core proteins in virus infection.

We have investigated the role of poly(ADP)-ribosylation of adenoviral proteins in virus infection. Viral core proteins V and the precursor to protein VII were shown to be in vivo and in vitro acceptors of ADP-ribose. In vivo ADP-ribosylation was restricted to viral proteins as the histones were not labeled during the late phase of infection. The ADP-ribosylated core proteins were assembled into mature virus particles. In vitro ADP-ribosylation of adenoviral core proteins performed with purified poly(ADP-ribose) polymerase led to relaxation of the chromatin structure of both ts1 and wild type pyridine cores and pentonless particles and triggered the complete dissociation of wild type particles. A critical role for poly(ADP)-ribosylation in virus infection was confirmed by measuring the effect of the inhibitors 3-aminobenzamide and nicotinamide on virus particle yield and infectivity. Both inhibitors depressed particle yield by up to 9-fold, but infectivity was reduced by up to 10(4)-fold. These results suggest that ADP-ribosylation of adenovirus core proteins may have a role in virus decapsidation.

Role of the nuclear matrix in adenovirus maturation.

The nuclear matrix has been implicated in several important cellular processes. In this paper, we investigate the role of the nuclear matrix in adenovirus type 2 assembly. Electron microscopic examination of nuclear matrices isolated from adenovirus infected Hep-2 cells clearly reveals that late in the lytic cycle, adenovirus capsids are intimately associated with the nuclear matrix. SDS-PAGE analysis showed that the viral core polypeptides V, PVII and 11 kDa were enriched in the nuclear matrix fraction. After a 3 h chase period a constant high ratio of PVII to VII prevailed in the nuclear matrix suggesting that mostly young virions and viral cores are bound to this structure. Most of the virus maturation endoproteinase activity co-purified with the nuclear matrix and the data suggest that the enzyme may be released from fragile young virions or assembly intermediates. Together these experiments suggest that the nuclear matrix is the site of adenovirus assembly and that mature virions may be released from the matrix by the viral endoproteinase.

The structure of adenovirus chromatin in infected cells.

The structure of adenovirus chromatin in infected cells was studied by micrococcal nuclease digestion and hybridization with virus-specific probes. In the early phase of infection (5 h) a significant proportion of viral molecules was organized like actively transcribed cellular chromatin. As expected for a transcriptionally active population of molecules, even at high multiplicity of infection the nucleosomal repeating pattern was less distinct than in a transformed cell which contained the corresponding but less active genomic region. The observed repeating pattern in infected cells was unlikely to be due to integrated molecules since less than 0.07% of input genomes became associated with cellular DNA. After the onset of viral DNA replication, the pool of viral chromatin organized like cellular chromatin rapidly increased. In addition, newly replicated molecules also maintained the cellular chromatin-like organization as measured by [3H]thymidine incorporation after the cessation of cellular DNA synthesis. These data suggest that newly replicated viral molecules are organized by histones into cell-like chromatin throughout the infection cycle. Coincident with the peak of viral DNA and core protein synthesis, and the decline of histone synthesis, the late, core-like non-repeating viral chromatin became dominant, increasingly obscuring the underlying repeating pattern. Experiments suggest that this late chromatin is destined for encapsidation, that the early chromatin persists and that viral core proteins do not displace histones on viral DNA. A model is proposed suggesting that transcription and type I replication occur on histone-condensed templates, while type II replication products late in infection are condensed by core proteins and are destined for encapsidation.

Unique sequences are interspersed among tandemly repeated elements in the murine gamma 1 switch segment.

Early in its differentiative pathway, a given B lymphocyte expresses immunoglobulin of the mu heavy chain class (IgM). Subsequent differentiative processes may involve rearrangement within the immunoglobulin heavy chain chromosomal locus to enable cells of the same lineage to synthesize immunoglobulins of other heavy chain classes (e. g. IgG, IgE or IgA), but with specificity for the same antigen as the original IgM molecule. Switch recombination, the molecular event which facilitates this chromosomal rearrangement, has been shown to occur between segments of DNA consisting of tandemly repeated unit sequences. These DNA segments have been functionally defined as switch regions. We have cloned the gamma 1 switch region from the BALB/c germline, and have demonstrated that significantly divergent sequence elements are interspersed among the tandemly repeated units characteristic of this switch region. We show that these unique elements exist in at least three copies within the switch segment, and discuss the implications of this novel and previously unreported primary structure.