Visualization of viral clearance in the living animal.

The early events in viral dissemination via the bloodstream were identified by monitoring the fate of 123I-radiolabeled reovirus after it was injected intravenously in rats. Continuous scintillation camera imaging showed that reovirus serotypes 1 and 3 were cleared from the circulation in less than 10 minutes by specific and distinct target organs. Reovirus serotype 1 accumulated predominantly in the lungs and the liver, whereas serotype 3 accumulated in the liver and the spleen with very little virus uptake by the lungs. Incubation of reovirus serotype 1 with a monoclonal antibody directed against the viral hemagglutinin before injection totally inhibited the clearance of the virus by the lungs. Similar results were obtained when viruses biolabeled with 35S were used. These results demonstrate that viruses can be rapidly transported through the bloodstream to specific target organs and that the localization of the viruses depends on the interaction between specific viral surface components and the target organ.

Identification and characterization of mutants affecting transcription termination at the threonine operon attenuator.

Mutations that map in or delete the attenuator of the threonine (thr) operon of Escherichia coli were isolated and characterized. These mutations disrupt or delete the transcription termination structure encoded by the attenuator leading to increased transcriptional readthrough into the thr operon structural genes. Most of the base substitutions and single base-pair insertions and deletions map in the G + C-rich region of dyad symmetry in the attenuator and decrease the calculated stabilities of the attenuator RNA secondary structures to similar extents (from -30.8 kcal/mol to approximately -21 kcal/mol). Most of the mutants showed a three- to fourfold increase in homoserine dehydrogenase (thrA gene product) synthesis relative to the wild-type parent strain. The mutation in one mutant (thrL153 + G) lowered the calculated stability of the RNA secondary structure only slightly (from -30.8 to 27.8 kcal/mol) but the mutant still exhibited high levels of homoserine dehydrogenase synthesis. In addition, three base substitution mutants (thrL135U, thrL139A and thrL156U) showed only slightly (1.5 to 2-fold) elevated levels of homoserine dehydrogenase activity, even though the calculated stabilities of the attenuator RNA secondary structures were reduced as much as most of the other mutants. Two of the mutations (thrL135U and thrL156U) mapped in the G + C-rich-A + T-rich junction of the attenuator. The third mutation (thrL139A) creates an A X C pair in the center of the G + C-rich region of the attenuator stem. The results obtained for these mutants show that the stability of the RNA secondary structure does not always correlate with the efficiency of transcription termination. Finally, analysis of the base changes in the substitution mutations showed that the mutational changes do not appear to be random.

Specificity of the attenuation response of the threonine operon of Escherichia coli is determined by the threonine and isoleucine codons in the leader transcript.

Expression of the threonine (thr) operon enzymes of Escherichia coli is regulated by an attenuation mechanism. The regulatory portion of the operon contains a region coding for a leader peptide that contains consecutive threonine and isoleucine codons. It is thought that translation of the leader peptide controls the frequency of transcription termination at the attenuator site. Using oligonucleotide-directed site-specific mutagenesis we have altered the putative control codons of the leader peptide coding region. In two of the mutants the threonine and isoleucine codons were changed to produce peptides containing histidine and tyrosine codons. Both mutants showed loss of regulation by threonine and isoleucine. A hisT mutation, which leads to an undermodification of tRNA(His), increased thr operon expression in the mutants threefold but did not affect expression of the wild-type thr operon. Two other mutants were constructed that contained two histidine codons early in the leader peptide. Expression in both of these mutants was unaltered by the presence of the hisT allele or by the addition of threonine and isoleucine to the growth medium. In addition, a wild-type strain containing a temperature-sensitive threonyl-tRNA synthetase mutation showed increased thr operon expression at the non-permissive temperature, whereas none of the mutants showed any change. Taken together these data indicate that the specificity of the attenuation response is effected by specific control codons within the thr leader peptide coding region. We have also directly demonstrated thr leader peptide synthesis in vitro using a plasmid encoding the wild-type thr leader region to direct the synthesis of a peptide of the appropriate molecular weight when labeled with [3H]threonine but not with [3H]histidine or [3H]tyrosine. Conversely, when extracts were incubated with templates containing the mutated DNAs, peptides were labeled that showed patterns consistent with the expected amino acid compositions. These data indicate that the thr leader RNA is translated into the predicted leader peptide.

A genetic enrichment for mutations constructed by oligodeoxynucleotide-directed mutagenesis.

A genetic enrichment procedure for mutations constructed by oligodeoxynucleotide(oligo)-directed mutagenesis of DNA cloned in M13mp vectors is described. The procedure uses an M13 vector that contains the cloned target DNA and amber (am) mutations within the phage genes I and II. This vector cannot replicate in a suppressor-free (sup degrees) bacterial strain. A gapped heteroduplex is formed by annealing portions of a complementary (-)strand containing wild-type copies of genes I and II to the am-containing template (+)strand. The oligo is annealed to the single-stranded (ss) region and the remaining gaps and nicks are repaired enzymatically to form a closed circular heteroduplex structure. By transfecting the DNA into a sup degrees host we promote the propagation of heteroduplexes with the oligo-containing (-)strand since only this construction contains the wild-type copies of genes I and II. This procedure eliminates the need for any physical separation of the covalently closed circular DNA that contains the oligo from the ss template. Using this technique we have constructed 17 point mutations with mutation frequencies ranging from 2-20% for single base changes and from 0.3-9% for multiple base changes. In addition, we found that the mutation frequencies were affected by the state of DNA methylation in the (+) and (-)strands.

Attenuation regulation in the thr operon of Escherichia coli K-12: molecular cloning and transcription of the controlling region.

Recombinant plasmids were constructed which carry defined regions of the threonine (thr) operon regulatory region of Escherichia coli. In vitro transcription experiments utilizing plasmid or restriction fragment templates showed that two major RNA transcripts, which differ in length by one to a few bases, are transcribed from this region. The approximate length of the transcripts is 150 to 170 bases, and the site(s) of termination is near or within the thr attenuator. The efficiency of termination at the thr operon attenuator in vitro is approximately 90%. A regulatory mutation, thr79-20, which is a G-C insertion in the attenuator, reduces the frequency of transcription termination to 75%. In addition, in vivo RNA transcripts were identified which hybridize to the thr operon regulatory region. These transcripts appeared to be identical to the two major in vitro transcripts as judged by their mobilities on 8% polyacrylamide-8 M urea gels. This result indicates that the thr operon regulatory region is transcribed in vivo and that termination occurs near or within the thr attenuator.

Contributions of RNA secondary structure and length of the thymidine tract to transcription termination at the thr operon attenuator.

The thr operon of Escherichia coli is regulated by an attenuation mechanism in which regulated transcription termination occurs in response to the levels of charged tRNAthr and tRNAile. Transcription of the thr operon regulatory region in vitro produces a 162-base transcript that is terminated efficiently at the attenuator. The attenuator sequence is similar to other rho-independent terminators. It contains a G + C region of dyad symmetry followed by a run of 9 A + T residues. We have characterized in detail the sequence requirements for efficient transcription termination in vitro. Using a set of point mutations in the G + C region of dyad symmetry of the thr attenuator, we have characterized the effects of these mutations on the efficiency of transcription termination. The efficiency was reduced in all of the mutants analyzed with the greatest effect being an approximate 20% decrease in termination. In some instances the electrophoretic mobilities of the terminated transcripts on 8% polyacrylamide, 8 M urea gels were shifted substantially relative to the wild type-terminated transcript, but the sites of transcription termination were altered by only a few base pairs. We also constructed a set of deletions removing consecutive thymidines which follow the G + C-rich region of dyad symmetry. Removal of 1 or 3 of the 9 thymidine residues had no effect on termination efficiency in vitro or in vivo. Removal of four to six thymidines caused a linear decrease in the efficiency of termination. When only one or two thymidines were present in the template, termination was completely abolished. These results indicate that both the integrity of the RNA stem and the length of the consecutive thymidine residues are important signals recognized by RNA polymerase during transcription of the thr operon regulatory region.

Use of randomly cloned DNA fragments for identification of Bacteroides thetaiotaomicron.

Randomly cloned fragments of DNA from Bacteroides thetaiotaomicron were used as hybridization probes for differentiation of B. thetaiotaomicron from closely related Bacteroides species. HindIII digestion fragments of DNA from B. thetaiotaomicron (type strain) were inserted into plasmid pBR322 and labeled with [alpha-32P]dCTP by nick translation. These labeled plasmids were screened for hybridization to HindIII digests of chromosomal DNA from type strains of the following human colonic Bacteroides species: B. thetaiotaomicron, Bacteroides ovatus, reference strain 3452-A (formerly part of B. distasonis), Bacteroides uniformis, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides distasonis, Bacteroides eggerthii, and reference strain B5-21 (formerly B. fragilis subsp. a). Two of the five cloned fragments hybridized only to DNA from B. thetaiotaomicron. Each of these two fragments hybridized to the same DNA restriction fragment in five strains of B. thetaiotaomicron other than the strain from which the DNA was cloned. One of the cloned fragments (pBT2) was further tested for specificity by determining its ability to hybridize to DNA from 65 additional strains of colonic Bacteroides.

RsaI: a new sequence-specific endonuclease activity from Rhodopseudomonas sphaeroides.

A new type II sequence-specific endonuclease, RsaI, has been identified from Rhodopseudomonas sphaeroides strain 28/5. An RsaI purification scheme that yields enzyme which is free of contaminating exonuclease and phosphatase activities after a single column fractionation has been developed. The enzyme recognized the tetranucleotide sequence 5'-GTAC-3' and cleaved between the T and A, thereby generating flush ends. RsaI should be extremely useful in deoxyribonucleic acid sequencing experiments.

Specificity and kinetics defining the interaction between a murine monoclonal autoantibody and DNA.

Interactions between a murine monoclonal anti-DNA autoantibody (BV17-45) and DNA were examined by direct binding and competitive radioimmunoassays. Binding isotherms constructed by titration of purified BV17-45 with a series of distinct 32P-labeled double-stranded DNA ([32P]dsDNA) fragments were super-impossible, suggesting: 1) BV17-45/[32P]dsDNA binding is independent of dsDNA size using fragments greater than or equal to 192 base pairs in length, and 2) BV17-45 does not exhibit stringent sequence specificity. Single-stranded DNA-specific monoclonal antibody BV04-01 did not react with [32P]dsDNA, confirming its duplex character. In competition experiments, BV17-45 cross-reacted with phage (phi X174, M13) RF AND VIRION DNAS AT PICOMOLAR concentrations. Selectivity for B-form DNA was suggested by the ability of poly(dA) . poly(dT), but not other helical duplex forms, to block BV17-45/[32P] dsDNA binding. Among the four deoxyribohomopolymers, only deoxyadenylic acid polymers completely inhibited BV17-45/[32P]dsDNA complex formation. [32P]dsDNA binding was relatively insensitive to ionic strength, suggesting minimal contribution of electrostatic forces to the binding free energy. Measured BV17-45/[32P]dsDNA association and dissociation rate constants (4 degrees C) were 7.4 X 10(6) M-1 s-1 and 9.2 X 10(-5) s-1, respectively, yielding a functional affinity of 8 X 10(10) M-1. Results are discussed in terms of the relative contribution of B-DNA structural and substructural determinants to the mechanism of BV17-45 recognition.

Structure and physical map of Rhodopseudomonas sphaeroides bacteriophage RS1 DNA.

We analyzed, by restriction endonuclease mapping and electron microscopy, the genome of the lytic Rhodopseudomonas sphaeroides-specific bacteriophage RS1 and characterized it as a linear molecule of approximately 60 to 65 kilobases. When the DNA from purified phage particles was examined by several independent methods, considerable size heterogeneity was apparent in the RS1 DNA. This size heterogeneity was concluded to be of biological origin, was independent of the specific host strain used to propagate virus, and was not due to the presence of host DNA within or nonspecifically associated with purified virions. In addition, treatment of RS1 DNA with either BAL 31 nuclease or DNA polymerase I Klenow fragment revealed that several distinct regions exist within the viral chromosome which contain free 3' hydroxyl groups. A restriction endonuclease map of the RS1 genome was constructed by using the restriction endonucleases EcoRI, ClaI, KpnI, BamHI, MluI, SmaI, and BclI; thereby allowing the positioning of some 40 restriction sites within the viral genome. The results are discussed in terms of the significance and the possible biological origin of the unique features discovered within the phage RS1 DNA.

Characterization of a site-specific restriction endonuclease from Rhodopseudomonas sphaeroides.

A type II restriction endonuclease, RshI, has been partially purified from photoheterotrophically grown Rhodopseudomonas sphaeroides strain 2.4.1. The enzyme preparation, after a single DE-52 column fractionation, is free of 5' exonuclease and phosphatase activities but contains a trace of 3' exonuclease activity. Based upon deoxyribonucleic acid (DNA) sequencing data in the vicinity of the enzyme-promoted cleavage of pBR322 DNA, we have concluded that RshI probably recognizes the palinodromic hexanucleotide sequence 5'-CGATCG-3' and cleaves between the T and C. lambda cI857 DNA contains three RshI sites, two of which lie in the replaceable region. The plasmid pBR322, which carries resistances to ampicillin and tetracycline, contains a single RshI site in the ampicillin resistance determinant. Insertion of DNA into the RshI site of pBR322 results in loss of ampicillin resistance but retention of tetracycline resistance, thereby providing a convenient screening procedure for recombinant plasmids.

Uptake of reovirus serotype 1 by the lungs from the bloodstream is mediated by the viral hemagglutinin.

We used the mammalian reoviruses to determine the molecular basis of the clearance of a virus from the bloodstream by specific organs. Reovirus serotypes 1 (T1) and 3 (T3) were radiolabeled with [35S]methionine or 125I, and the viruses were injected intravenously into weanling rats. The distribution of radioactivity within the animals was determined at various times after the injection. Both viruses were cleared rapidly from the bloodstream and concentrated in different organs. Reovirus T1 was found predominantly in the lungs and liver, whereas T3 was found predominantly in the liver, with very little virus in the lungs. Using intertypic reassortants, we determined that the T1 S1 gene, which encodes the viral hemagglutinin (sigma 1 protein), is responsible for the difference in uptake of T1 and T3 by the lungs. The genetic mapping was extended by using several approaches. (i) T1 subjected to limited proteolytic digestion with chymotrypsin was cleared efficiently by the lungs despite the removal of sigma 3 and digestion of mu 1C to delta. (ii) Uptake of T1 by the lungs was totally inhibited by incubation of T1 with an anti-sigma 1 monoclonal antibody or its Fab fragment before injection. (iii) A reovirus T1 variant in the sigma 1 protein was poorly taken up by the lungs. These data indicate that clearance of reovirus from the bloodstream by the lungs is dependent on the presence of the T1 sigma 1 protein.