Escherichia coli

Scand J Gastroenterol Suppl, 1982, 77, 47 - 59
Pathogenic mechanisms and new perspectives in the treatment and prevention of enteric infections; Holmgren J et al.; PIP: Enteric infections cause more than 1 billion episodes of diarrheal disease in humans each year killing many millions of people, especially young children, in developing countries . Recent progress, reviewed here, has enabled that a specific pathogen can now be isolated in the majority of patients with acute diarrhea, and has also elucidated fundamental pathogenic mechanisms and their pathophysiological effects for several of these agents . Based on this understanding, it now seems possible to devise new techniques for the treatment and prevention of diarrheal disease to complement those based on fluid replacement therapy and sanitation . Prospects for the development of new or improved vaccines, receptor-prophylactic binding agents, and antisecretory drugs are discussed . author's modified

Mol Gen Genet, 1982, 186(3), 385 - 90
Cloning and restriction mapping of the yeast URA2 gene coding for the carbamyl phosphate synthetase aspartate-transcarbamylase complex; Souciet JL et al.; Two yeast DNA pools inserted in a hybrid Escherichia coli-yeast vector pFL1 were used to transform E . coli and yeast aspartate-transcarbamylase-less strains to prototrophy . From the first pool--a BamHI yeast DNA digest--a 6.4 kb BamHI fragment was recovered that gave good complementation of the E . coli auxotrophy but poor complementation of the yeast auxotrophy . From the second pool--a partial Sau3A yeast DNA digest--five independent plasmids complementing either E . coli, yeast, or both were recovered . Each of the five plasmids possessed sequences in common with the 6.4 kb BamHI fragment . One of these plasmids, which complemented the two URA2 activities in yeast and which produced a carbamyl-phosphate synthetase, aspartate-transcarbamylase complex sensitive to UTP feedback inhibition contained the full URA2 gene . A restriction map of the URA2 gene has been constructed and seven different consecutive segments have been recloned in pBR322 to measure their hybridization with URA2 messenger RNA, allowing us to estimate the limits of the gene.

J Interferon Res, 1982, 2(2), 151 - 7
Effect of murine beta-interferon preparation on phagocytosis and cyclic AMP levels in mouse peritoneal macrophages; Degre M et al.; Mouse peritoneal macrophages (MPM) cultivated with a mouse beta-interferon preparation (MuIFN-beta) or "mock" IFN were tested for phagocytic ability and intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) . Suspensions of nonopsonized Escherichia coli (E . coli) were used for phagocytosis experiments . Treatment of MPM with 10(1)-10(3) U per ml of MuIFN-beta stimulated the phagocytic activity and raised the levels of cAMP in MPM . The effect of MuIFN-beta on cAMP levels were dose and time dependent . Maximal cAMP levels were seen when MPM were incubated with 10(3)-10(4) U per ml of MuIFN-beta for five hours . Simultaneous addition of MuIFN-beta and the adenylcyclase inhibitor N-ethylmaleimide to the MPM cultures prevented the rise in cAMP levels but not the increased phagocytic capacity . MuIFN-beta induced enhancement of phagocytosis and elevation of cAMP levels in MPM seem to be two parallel but not interlinked processes.

Basic Life Sci, 1982, 20, 245 - 58
Mutants of Escherichia coli K12 which affect excision of transposon Tn10; Lundblad V et al.; We have described three illegitimate recombination events associated with, but not promoted by, transposon Tn10: precise excision, nearly precise excision, and precise excision of a nearly precise excision remnant . All three are structurally analogous: excision occurs between two short direct repeat sequences, removing all intervening material plus one copy of the direct repeat . In each case, the direct repeats border a larger inverted repeat . We report here the isolation of host mutants of Escherichia coli K12 which exhibit increased frequencies of precise excision of Tn10 . Nineteen of the 39 mutants have been mapped to five distinct loci on the E . coli genetic map and have been designated texA through texE (for Tn10 excision) . Mapping and genetic characterization indicate that each tex gene corresponds to a previously identified gene involved in cellular DNA metabolism: recB and/or recC, uvrD, mutH, mutS, and dam . The role of these various DNA repair and recombination genes in an illegitimate recombination process such as Tn10 excision will be discussed . In addition to an increase in precise excision frequency, all 39 tex mutants display an increased frequency for nearly precise excision . However, none of the mutants are increased for the third excision event, precise excision of a nearly precise excision remnant, supporting the idea that precise and nearly precise excision occur by closely related pathways which are distinct from those pathways which promote the third type of excision event.

Basic Life Sci, 1982, 20, 235 - 44
Low level and high level DNA rearrangements in Escherichia coli; Bukhari AI et al.; It can be argued that all organisms exhibit two levels of DNA rearrangements . At a low level they may occur sporadically in cells, perhaps largely because of spontaneous activity of transposable genetic elements . A high level may be induced in special circumstances if functions that cause rearrangements are hyperactive . As an example of low level genetic rearrangements, we have studied the occurrence of spontaneous polar mutations in the early regions of prophage Mu . We isolated 49 independent prophage mutants, which are defective in replication ad expression of late genes; 44 were in the B region and 5 were in the A region . In the B region, 68% were IS1 insertions, 9% were IS5 insertions and 9% were IS2 insertions; 14% showed no insertion . In the A region, all 5 were IS5 insertions . Thus most spontaneous polar mutations in Escherichia coli appear to the insertions . IS1 is the most common insertion; however, certain DNA rearrangements are exemplified by DNA fusion and DNA dissociation that occur when replication-transposition functions of Mu are induced.

Z Allg Mikrobiol, 1982, 22(3), 211 - 4
Regulation of bistability in glucose metabolism of Escherichia coli ML 30 chemostat cultures by cyclic AMP; Muller PJ et al.; Evidence is presented that cyclic AMP is engaged in the regulation of a bistability in the glucose and energy metabolism of NH3-limited chemostat cultures of Escherichia coli ML 30 . Cyclic AMP probably reverses the repression of the citric acid cycle by glucose favouring the state of glycogen and energy overproduction.

Mol Cell Biol, 1982 Jan, 2(1), 88 - 92
Syntheses and stabilities of proteins related to the polyoma small T antigen in Escherichia coli; Horwich A et al.; We compared the syntheses and turnovers of two proteins related to the polyoma small T antigen synthesized in Escherichia coli from plasmids containing polyoma genomic segments joined to lac control elements . A protein with an authentic polyoma N terminus was more unstable than a protein with N-terminal amino acids derived from beta-galactosidase . Both were more unstable than most bacterial proteins.

Mol Gen Genet, 1982, 186(1), 87 - 94
Continuous synthesis of the dnaA gene product of Escherichia coli in the cell cycle; Sakakibara Y et al.; The dnaA gene product of Escherichia coli, identified as a weakly basic protein of about 48,000 daltons (Yuasa and Sakakibara 1980), can be separated from other cellular proteins by means of two-dimensional gel electrophoresis . Synthesis of the dnaA protein took place continuously during a cell growth cycle . The newly synthesized dnaA protein persisted stably for one generation . Thermosensitive dnaA protein produced by the dnaA167 mutant was stable at 30 degrees C, but was disintegrated at 42 degrees C . The amount of intact dnaA protein present in the mutant exposed to the high temperature for 60 min was less than a quarter of the amount at the time of the shift . The cells having the reduced amount of intact dnaA protein were capable of initiating a new round of chromosome replication at the low temperature without de novo synthesis of the dnaA protein . The potential of the mutant for initiation of DNA replication decreased with reduction in the amount of the thermoreversible dnaA protein . The mutations dnaA167 and dnaA46 had no significant effect on the syntheses of the dnaA mRNA and the protein product at the low and high temperatures.

Mol Gen Genet, 1982, 186(1), 71 - 7
Regulation of DNA synthesis and capacity for initiation in DNA temperature mutants of Escherichia coli . III . Synthesis of the dnaA protein and of DNA-binding proteins; Eberle H et al.; The synthesis and action of the dnaA product with respect to DNA initiation and the synthesis of DNA-binding proteins in Escherichia coli was examined . Results indicate that when dnaA product is irreversibly denatured and must be synthesized before initiation can occur, its synthesis and action appear to be complete approximately 30 min before initiation takes place . However, in mutants whose dnaA product is temperature reversible the action of the dnaA product appears to occur near the time of initiation . Examination of the DNA-binding proteins from the mutants suggests that a 53 kd protein, possibly the dnaA product, may be synthesized at the time of initiation under normal conditions at permissive temperature . The presence of active dnaA product appears to trigger the synthesis of a 60-65 kd protein which may be responsible for preventing another immediate initiation event.

Mol Gen Genet, 1982, 186(1), 145 - 52
Cloning and expression in Escherichia coli K-12 of the biosynthetic dehydroquinase function of the arom cluster gene from the eucaryote, Aspergillus nidulans; Kinghorn JR et al.; A 1.35 Md DNA HindIII fragment containing part of the arom gene cluster or cluster gene of Aspergillus nidulans encoding biosynthetic dehydroquinase (5-dehydroquinate hydrolyase) has been cloned in plasmid pBR322 on the basis of functional expression in Escherichia coli . The fungal fragment on pBR322, designated pHK29, complements a corresponding E . coli dehydroquinase structural gene (aroD) mutation . pHK29 contains one BamHI, HpaII, PstI, SmaI, XhoI and surprisingly, one HindIII site since pHK29 hybrid Aspergillus DNA is a HindIII fragment itself . The biosynthetic dehydroquinase activity extracted from E . coli strains, containing pHK29, had properties similar to those of the enzyme activity from Aspergillus . The protein specified by pHK29 appears to be 80 kd . No increase of dehydroquinase activity was found in polynucleotide phosphorylase deficient strains (pnp) of E . coli.

Mol Gen Genet, 1982, 186(1), 106 - 10
Reiterated DNA sequences in a mutant strain of Streptomyces glaucescens and cloning of the sequence in Escherichia coli; Ono H et al.; Streptomyces glaucescens exhibits a high degree of genetic instability . A sequence of 7.2 kb has been found which is present in a few tandemly repeated copies in the wild type strain GLA 0 and is amplified to ca . 500 copies per genome in the mutant strain GLA 1204 . This sequence was cloned in Escherichia coli using pBR325 as vector.

J Mol Appl Genet, 1982, 1(4), 289 - 99
Use of a lac promoter-operator fragment as a transcriptional control switch for expression of the constitutive lpp gene in Escherichia coli; Nakamura K et al.; We constructed hybrid plasmids to allow controlled expression of the lpp gene coding for the outer membrane lipoprotein of Escherichia coli, which is otherwise expressed constitutively . This was achieved by the insertion of a DNA fragment carrying the lacUV5 promoter-operator region as a transcriptional control switch into the 5'-untranslated region of the lpp gene . When fully induced, the production of the lipoprotein, controlled under the tandem promoters of lppp-lacpo-lpp, increased approximately 3-fold compared to that under lacpo-lpp control . However, it was still only one-third of the lipoprotein production under the constitutive lpp expression . One such plasmid, pKEN125, carrying lppp-lacpo-lpp in pBR322 produced only a trace amount of the lipoprotein without induction in an E . coli lpp- cell . Upon the addition of isopropyl-beta-d-thiogalactoside, however, the amount of the lipoprotein reached almost 40% of the total membrane proteins . Cells carrying pKEN125 grew normally in the presence of the inducer, whereas cells carrying plasmid pKEN126 with tandem duplication of lppp-lacpo-lpp sequences in pBR322 lysed upon induction at high temperature . In cells with pKEN126 induced at high temperature, at least three new bands which were cross-reactive with antilipoprotein serum in addition to the mature lipoprotein were detected by pulse-labeling cells with {35S}methionine.

Mol Gen Genet, 1982, 185(3), 515 - 7
The use of the plasmid pHA10 in the isolation of lambda PL promoter mutations; Hyman HC et al.; The plasmid pHA10 provides a good positive selection system for PL promoter mutations . It contains the lambda EcoR1-D fragment cloned into the pBR322 plasmid . Inactivation of the temperature sensitive lambda repressor at 42 degrees C leads to transcription of the kil gene and host death . One group of survivors at 42 degrees C is saved due to mutations in the PL promoter . After several screening procedures one such mutant was isolated . Restriction enzyme analysis of the plasmid DNA followed by sequence determination showed it to be a 24 base pair deletion beginning half-way through the "Pribnow box" continuing through part of the -35 region . It removes the OL1 section of the OL operator plus one base pair of OL2 . This mutation (called dpl) may be useful to the study of the lambda OLPL regulation system . The effect of base changes within the PL promoter on the promoter activity are discussed.

Mol Gen Genet, 1982, 185(3), 510 - 2
Recombinant plasmids with genes for the biosynthesis of alkaline phosphatase of Escherichia coli; Boidol W et al.; Restriction maps of several recombinant plasmids representing a section of the E . coli K12 chromosome 35,000 bp in size with the genes phoA, proC and phoB were prepared . The orientation of phoA and the exact position of its N-terminal end on this map were determined by identifying a subfragment which carried the phoA promoter and by determining the nucleotide sequence of a 160 bp portion of this subfragment comprising the codons for the N-terminal end of pre-alkaline phosphatase . From this DNA sequence the leader sequence of alkaline phosphatase which consists of 21 amino acids was derived.

Mol Gen Genet, 1982, 185(3), 493 - 7
Lysis of Escherichia coli by induction of cloned phi X174 genes; Henrich B et al.; The largest of the fragments produced by AluI digestion of phi X174 RFI DNA comprises genes E and J as well as parts of genes D and F . This DNA fragment (1007 bp) was cloned into the lac z' gene of plasmid pUR222 . In the recombinant plasmid pUH12, transcription of the phi X174 genes is controlled by the lac p-o region . Induction of the cloned genes by addition of the lac inducer, IPTG, resulted in lysis of the bacteria . Cloning of the corresponding AluI-fragment from phi X174am3 DNA, carrying an amber mutation in gene E, showed that the expression of this gene alone is sufficient to trigger cell lysis . The time interval between the addition of IPTG and the onset of lysis depended on the concentration of the inducer, however, the rate of lysis was similar at all IPTG concentrations used.

Mol Gen Genet, 1982, 185(3), 487 - 92
A secondary promoter for elongation factor Tu synthesis in the str ribosomal protein operon of Escherichia coli; Zengel JM et al.; The str operon of Escherichia coli contains genes for ribosomal proteins S12 and S7 and for elongation factors EF-G and EF-Tu (Jaskunas et al . 1975) . We have subcloned various segments of DNA from this operon onto multicopy plasmids . We found that cells carrying a recombinant plasmid which lacks the major promoter for the str operon but contains the 5' portion of the EF-Tu gene synthesize a novel protein which we have identified as a truncated EF-Tu molecule . Moreover, cells carrying plasmids with an intact EF-Tu gene synthesize the elongation factor at a 3- to 5-fold higher rate than haploid cells . Thus the EF-Tu gene can be expressed in the absence of the major promoter for the str operon . This expression is not due to read-through from plasmid promoters, but it is dependent on the presence of the distal portion of the EF-G gene on the plasmids . These results indicate that there is a secondary promoter for EF-Tu expression, apparently located within the structural gene for elongation factor EF-G.

Mol Gen Genet, 1982, 185(3), 408 - 17
Read-through transcription from a derepressed Tn3 promoter affects ColE1 functions on a ColE1::Tn3 composite plasmid; Emerick AW; Mutations in the repressor encoded by the transposon Tn3 tnpR gene lead to increased levels of expression of two gene products: the mutant repressor (TnpR-) and the Tn3 encoded transposase, TnpA (Heffron et al . 1978; Chou et al . 1979a) . Derivatives of the ColE1::Tn3 composite plasmid, RSF2124, with mutant Tn3 repressor exhibited the expected elevated levels of transposition . Unexpectedly, hosts containing these tnpR- derivatives produced enhanced levels of the ColE1 encoded toxin, colicin E1 . The gene for colicin E1 maps far (0.23-0.98 MU) from the Tn3 insertion point (0.73 MU) (Fig . 1) . The colicin E1 overproduction phenotype, designated Eop-, was complemented in trans by wild type repressor gene product (TnpR+) to the wild type phenotype, Eop+ . Hosts with RSF2124 derivatives which expressed high levels of both mutant repressor and mutant transposase (TnpR-, TnpA-) were Eop- . Hosts containing plasmids deleted for both tnpA and tnpR promoters were Eop+, while hosts with plasmids carrying a lac promoter substitution for the tnpA promoter were Eop- . These data support the idea that a cis-acting effect of increased transcription from the tnpA promoter into adjacent ColE1 DNA was the cause of colicin overproduction . Increased transcription activated a putative colicin augmentation function (caf) whose presence was required for the Eop- phenotype . Deletion mapping established that one boundary of the caf locus lies within 52 bases of the junction of the left end of Tn3 and ColE1 DNA . ColE1 DNA in this area contains an open reading frame which could encode either a 74 or a 63 residue protein (B . Polisky, unpublished DNA sequence data) . The presence of increased levels of an mRNA transcript from this region and/or the increased expression of protein(s) from this transcript could result in an Eop- phenotype . Expression of the Eop- phenotype requires the presence of the host recE gene . Evidence is presented which suggests that the recA repressor, lexA protein, controls expression of the recE gene product, ExoVIII.

Adv Exp Med Biol, 1982, 141, 453 - 61
Release of the membrane-calcium and its relation to the superoxide formation by polymorphonuclear leukocytes; Takeshige K et al.; The relationship between the intracellular translocation of calcium from the storage pool and the oxidative metabolism was studied . An intracellular calcium-antagonist, TMB-8, inhibited the release of superoxide induced by a calcium ionophore A23187 and the inhibition was relieved by the addition of calcium ions . The release induced by cytochalasin D or by the ingestion of bacteria was similarly inhibited by TMB-8 . The mobilization of intracellular divalent cations of leukocytes was monitored by a fluorescent probe, CTC . When the CTC-loaded cells were stimulated with cytochalasin D or E . coli, a fluorescence change ascribable to the release of calcium from the intracellular hydrophobic environment was observed . The dose-response curve of the fluorescence change and that of the superoxide release of th cells were very similar . TMB-8 inhibited both metabolic and fluorescence changes in parallel . The results support the hypothesis that an intracellular translocation of calcium is stimulated the oxidative metabolism of leukocytes.

Res Vet Sci, 1982 Jan, 32(1), 70 - 3
Diarrhoea in dairy calves reduced by feeding colostrum from cows vaccinated with rotavirus; Snodgrass DR et al.; Forty-two dairy calves remained with their dams for two days after birth, and then were removed to a calf rearing shed . Calves were allocated to three groups for the next 14 days, and received twice daily either whole milk, whole milk with a 10 per cent supplement of pooled normal bovine colostrum or whole milk with 10 per cent supplement of colostrum from cows vaccinated with rotavirus . A natural outbreak of diarrhoea occurred, affecting 28 of the 42 calves . Feeding immune colostrum delayed the onset of diarrhoea, and reduced its incidence, duration and severity . Live weight gains were consequently improved . The group fed normal colostrum had diarrhoea intermediate in severity between that of control calves and those fed immune colostrum . The aetiology of the diarrhoea was complex, with calves excreting rotavirus, enteropathogenic Escherichia coli and cryptosporidia.

Mol Gen Genet, 1982, 185(2), 262 - 8
Multiple regulation of the activity of adenylate cyclase in Escherichia coli; Joseph E et al.; We have studied the correlation between the activities of adenylate cyclase (ATP pyrophosphatelyase-(cyclizing); EC 4.6.1.1) and in vivo rates of synthesis and intracellular concentrations of adenosine 3',5' cyclic monophosphate (cAMP) under various growth conditions in wild-type Escherichia coli and in mutants lacking or overproducing the cAMP receptor protein (CAP) . We showed that when wild-type bacteria are grown in the presence of a variety of carbon sources the intracellular concentrations of cAMP are inversely related to the adenylate cyclase activities determined in permeabilized cells, suggesting that the carbon source-dependent modulation of cAMP levels is not directly related to the regulation of adenylate cyclase activity . In mutants lacking functional CAP (crp) the in vivo rates of cAMP synthesis are several hundred-fold higher than in the wild-type parent without a parallel increase of adenylate cyclase activities . In a strain carrying multiple copies of the crp gene and overproducing CAP the activity of adenylate cyclase is severely inhibited, although the in vivo rate of cAMP synthesis is similar to the parental strain . We interpret these results as indicating that CAP controls mainly the activity rather than the synthesis of adenylate cyclase.

Mol Gen Genet, 1982, 185(2), 223 - 8
pIJ101, a multi-copy broad host-range Streptomyces plasmid: functional analysis and development of DNA cloning vectors; Kieser T et al.; Streptomyces lividans ISP 5434 contains four small high copy number plasmids: pIJ101 (8.9 kb), pIJ102 (4.0 kb), pIJ103 (3.9 kb) and pIJ104 (4.9 kb) . The three smaller species appear to be naturally occurring deletion variants of pIJ101 . pIJ101 and its in vivo and in vitro derivatives were studied after transformation into S . lividans 66 . pIJ101 was found to be self-transmissible by conjugation, to elicit "lethal zygosis" and to promote chromosomal recombination at high frequency in both S . lividans 66 and S . coelicolor A3(2) . A restriction endonuclease cleavage map of pIJ101 was constructed for 11 endonucleases; sites for five others were lacking . Many variants of pIJ101 were constructed in vitro by inserting DNA fragments determining resistance to neomycin, thiostrepton or viomycin, and having BamHI termini, into MboI or BclI sites on the plasmid, sometimes with deletion of segments of plasmid DNA . The physical maps of these plasmids were related to their phenotypes in respect of lethal zygosis and transfer properties . In vivo recombination tests between pairs of variant plasmids were also done . These physical and genetic studies indicated that determinants of conjugal transfer occupy less than 2.1 kb of the plasmid . A second segment is required for spread of the plasmid within a plasmid-free culture to produce the normal lethal zygosis phenotype: insertion of foreign DNA in this region caused a marked reduction in the diameter of lethal zygosis zones . The minimum replicon was deduced to be 2.1 kb or less in size; adjacent to this region is a 0.5 kb segment which may be required for stable inheritance of the plasmid . The copy number of several derivatives of pIJ101 in S . lividans 66 was between 40 and 300 per chromosome and appeared to vary with the age or physiological state of the culture . pIJ101 derivatives have a wide host range within the genus Streptomyces: 13 out of 18 strains, of diverse species, were successfully transformed . Knowledge of dispensable DNA segments and the availability of restriction sites for the insertion of DNA, deduced from the properties of plasmids carrying the E . coli plasmid pACYC184 introduced at various sites, was used in the construction of several derivatives of pIJ101 suitable as DNA cloning vectors . These were mostly designed to be non-conjugative and to carry pairs of resistance genes for selection . They include a bifunctional shuttle vector for E . coli and Streptomyces; a Streptomyces viomycin resistance gene of this plasmid is expressed in both hosts.

Mol Gen Genet, 1982, 185(2), 216 - 22
Bidirectional deletions associated with IS4; Habermann P et al.; A new type of chromosomal rearrangements associated with a transposable element has been described for IS4 . These rearrangements are deletions, in which the transposable element IS4 and DNA adjacent to it on either side is lost . These deletions are at least ten times more frequent in the presence of IS4, than when the element is absent from that region . The formation of bidirectional deletions is more than 1,000 times more frequent than precise excision of IS4.

Mol Gen Genet, 1982, 185(1), 88 - 92
A DNA sequence containing the control sites for gene malT and for the malPQ operon; Debarbouille M et al.; The order of 802 base pairs was established in a DNA segment containing the promoter for malPQ which is one of the three maltose operons, and the promoter for malT, the positive regulator gene of the maltose regulon . The determination of the amino-terminal sequence of the MalT protein allowed us to identify the beginning of the malT gene on the sequence . The position of the malP gene was deduced from the published amino-terminal sequence of maltodextrin phosphorylase . A total of 611 base pairs separate the initiation codons for these two genes, which are transcribed in opposite directions . This large intergenic region does not code for any polypeptide of significant size . The main features of this sequence are discussed in terms of the regulation known to operate on malT and malPQ expression.

Mol Gen Genet, 1982, 185(1), 82 - 7
A DNA sequence containing the control regions of the malEFG and malK-lamB operons in Escherichia coli K12; Bedouelle H et al.; The malB region in E . coli is composed of two operons, malEFG and malK-lamB, transcribed divergently from a control region located between the malE and malK genes . We have established the DNA sequence of a 600 base pair segment which contains the sites necessary for initiation of transcription of the malB operons and the translation start sites for malE and malK . We have also determined the position of a 147 base pair deletion, malB delta 100, in this sequence . The control region contains two short segments (about 35 base pairs) showing unusual distributions of bases: the GC content (85%) is higher than average and most of the purines (85%) are on one strand . We discuss the possible relevance of this and other salient features of the DNA sequence to the mechanisms by which the expression of the malB region is positively regulated.

Mol Gen Genet, 1982, 185(1), 169 - 75
Recombination between two TnA transposon sequences oriented as inverse repeats is found less frequently than between direct repeats; Chiang SJ et al.; Inverse repeats of the transposon Tn2660 in either a ColE1 or an R6K replicon, with or without inversions of the parental DNA sequences between the repeats, show no detectable (less than 2%) evidence of recombination between the repeats after 60 generations of growth in either recA or RecA+ hosts . In contrast, attempts made to construct plasmids which carry two direct repeats by in vitro cleavage and ligation in the recA host were unsuccessful, although homologous plasmids with inverse repeats could be constructed, and other plasmids were found consistent with products of recombination between the direct repeats of the transient intermediate structure . It is concluded that in recA or recA+ hosts recombination between direct repeats of a transposon is frequent, whereas recombination between inverse repeats of a homologous structure has not been observed . A model to explain this difference depends upon a mechanisms that produces a nick in only one of the pair of strands at the internal resolution site (IRS) sequence of the transposon.

Mol Gen Genet, 1982, 185(1), 142 - 7
Genetic mapping of mutation in Escherichia coli leading to a temperature-sensitive RNase D; Zaniewski R et al.; In order to determine the metabolic role of RNase D in Escherichia coli, we have attempted to isolate strains deficient in this enzyme . One strain containing a temperature-sensitive RNase D was found among a heavily mutagenized stock of strain temperature-sensitive for growth . Genetic mapping of the mutation responsible for the altered RNAse D enabled us to define the rnd locus, at 39.5-40.0 min on the E . coli map, which apparently specifies the RNase D structural gene . Using a Tn10 insertion near the rnd locus, we constructed isogenic strains containing RNase D and Rnase II mutations, alone or in combination . Although the original mutant isolate displayed temperature-sensitive growth . no growth phenotype was associated with the rnd mutation in wild type background, possibly because a substantial amount of RNase D remained in cells grown at 45 degrees C . However, elucidation of the map position of the rnd locus should prove useful for the isolation of other mutant strains with lower levels of RNase D.

J Gen Microbiol, 1982 Jan, 128 (Pt 1), 219 - 22
Generation of a membrane potential by one of two independent pathways for nitrite reduction by Escherichia coli; Pope NR et al.; A set of four isogenic Escherichia coli strains has been constructed in which all possible combinations of NADH- and formate-dependent nitrite reductases are active or inactive . Each pathway can be inactivated genetically without a corresponding loss in the other activity: the two pathways are therefore biochemically independent . The generation of a membrane potential during nitrite reduction by formate has been demonstrated using an ion-selective electrode specific for a lipophilic cation . The observed energy conservation results, at least in part, from the ability of formate dehydrogenase in E . coli to pump protons.

Aust Vet J, 1982 Jan, 58(1), 20 - 3
Enteritis in foals induced by rotavirus and enterotoxigenic Escherichia coli; Tzipori S et al.; Colostrum-deprived, colostrum-fed or suckling foals were orally inoculated with foal rotavirus and enterotoxigenic Escherichia coli derived from a calf . Neither agent given alone caused diarrhoea in foals aged 1 or 2 days, although with rotavirus, 2 of the 3 inoculated foals became depressed 3 days after inoculation and all 3 were excreting rotavirus in the faeces . Inoculation of both agents induced diarrhoea in colostrum-deprived, colostrum-fed or suckling foals aged up to 16 days . There was an apparent age-related resistance to diarrhoea which developed between 2 and 3 weeks of age . It was related to failure of rotavirus to establish apparent infection in older foals and was independent of preinoculation maternal antibody.

Proc Natl Acad Sci U S A, 1982 Jan, 79(2), 330 - 4
Isolation of an altered form of DNA polymerase I from Escherichia coli cells induced for recA/lexA functions; Lackey D et al.; A novel form of DNA polymerase I (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, DNA nucleotidyltransferase, EC 2.7.7.7) activity has been isolated from Escherichia coli cells that had been activated for expression of the DNA damage-inducible genes . Induction was by treatment of normal cells or cells carrying the spr-51 and tif-1 mutations with nalidixic acid . This activity, DNA polymerase I, seems to be a form of DNA polymerase I because it is insensitive to N-ethylmaleimide, is inhibited by antibody to DNA polymerase I, and does not appear in a polA1 strain . DNA polymerase I activity sediments through sucrose gradients as a broad peak with s20.w = 6.6--10.5, compared with an s20,w = 4.8--5.5 for DNA polymerase I . The fidelity during polymerization reactions of DNA polymerase I is relatively low with a variety of synthetic templates and deoxynucleoside triphosphates, although the enzyme appears to have a normal level of 3' greater than 5' exonuclease . Polymerase I has properties that might implicate it in some form of mutagenic DNA repair.

Proc Natl Acad Sci U S A, 1982 Jan, 79(2), 320 - 4
Gene order and gene-polypeptide relationships of the proton-translocating ATPase operon (unc) of Escherichia coli; Gunsalus RP et al.; We have constructed an extensive set of plasmids that carry the genes specifying the eight polypeptides of the proton-translocating ATPase of Escherichia coli . Using detailed restriction analysis and in vitro protein synthesis directed by these plasmids, we have established the order of the eight unc genes to be BEFHAGDC and the corresponding polypeptides to be a, c, b, delta, alpha, gamma, beta, and epsilon . These analyses include determining the location of the gene coding for the delta subunit of the F1 portion of the complex . We call this gene uncH . We have now established the gene order and gene-polypeptide relationships of the unc operon . This approach should be of use for study of other multigene bacterial operons, especially those with genes coding for polypeptides with unknown or unmeasurable catalytic activity.

Proc Natl Acad Sci U S A, 1982 Jan, 79(2), 257 - 61
Efficient expression of Escherichia coli galactokinase gene in mammalian cells; Schumperli D et al.; The Escherichia coli galactokinase gene (galK) was inserted into a modified early region transcription unit of simian virus 40 (SV40) contained on a bacterial plasmid . Introduction of this pSVK vector into monkey, mouse, and hamster cell lines by transfection resulted in efficient expression of the bacterial galK gene . This expression was shown to be dependent upon fusion of the galK gene to the early promoter of SV40 and did not appear to require SV40 splice signals . Moreover, expression in these cells could be obtained either transiently, 24--72 hr after transfection, or continuously, after stable transformation . In particular, pSVK-dependent galK expression was obtained in a hamster cell line genetically deficient in galactokinase activity . Expression of the bacterial enzyme was shown to complement the galactosemic defect of these cells, thereby allowing their selective survival and growth on galactose as the only carbon source . The ability to readily assay, select for, and potentially select against galK expression from pSVK and its derivatives should prove extremely useful in studying eukaryotic gene regulatory signals.

Proc Natl Acad Sci U S A, 1982 Jan, 79(2), 233 - 7
Human influenza virus hemagglutinin is expressed in monkey cells using simian virus 40 vectors; Hartman JR et al.; We have cloned and expressed the hemagglutinin (HA) gene of a human influenza virus (A/WSN/33) in monkey kidney cells by linking it to deleted simian virus 40 (SV40) genomes that contain the entire early gene region, the origin of replication, and late leader sequences . The HA gene (1775 base pairs long) was originally inserted by the dG . dC tailing technique into the multicopy plasmid of Escherichia coli, pBR322, using cDNA made from viral RNA . The cloned gene was further modified by treatment with nuclease Bal 31 to remove the dG . dC tails and some of the untranslated sequences and recloned in E . coli after addition of BamHI restriction endonuclease linkers . A number of SV40 and HA recombinants (SV--HA) were constructed by inserting recloned HA DNA into the late gene region of SV40 . The SV--HA recombinants, when complemented in a lytic infection of monkey cells by the helper function of SV40 early deletion mutants expressed influenza HA as detected by immunofluorescence and immunoprecipitation of in vivo-labeled proteins using either heterogeneous anti-influenza rabbit antibodies or monoclonal antibodies against HA . Furthermore, the WSN HA expressed by the SV--HA recombinants was also glycosylated and possessed the same molecular weight (approximately 70,000) as the uncleaved HA of WSN virus in monkey cells.

Gene, 1982 Jan, 17(1), 9 - 18
Cloning of small DNA fragments containing the Escherichia coli tryptophan operon promoter and operator; Russel DR et al.; A41-bp AluI restriction fragment from the trp promoter-operator region has been cloned into the PvuII site of pBR322, regenerating PvuII sites . Transformants were selected on media that allowed the selection of trp-operator-bearing plasmids . The cloned 41-bp fragment can be released from the vector by PvuII digestion, and it possesses a functional promoter and operator as demonstrated by in vivo tests . The 41-bp fragment contains several restriction sites: HincII, TaqI, RsaI, and a HpaI site that is located at the center of the operator sequence . Two new operator derivatives, symmetrical about the HpaI site, were prepared from the 41-bp fragment by joining two right-side, or two left-side PvuII-HpaI pieces together at the HpaI site . These derivatives showed in vivo operator activity . Plasmids containing up to five copies of the 41-bp trp-promoter-operator fragment have been constructed . These plasmids should be useful in preparing large amounts of the 41-bp fragment.

Gene, 1982 Jan, 17(1), 79 - 89
Construction and characterization of new cloning vehicles . VI . Plasmid pBR329, a new derivative of pBR328 lacking the 482-base-pair inverted duplication; Covarrubias L et al.; The 4150-bp plasmid pBR329 was constructed by the the insertion into pBR327 of an 877-bp DNA fragment carrying the Cmr gene from pBR328 . This new cloning vector does not contain the 482-bp inverted duplication that has been reported to be present in pBR325 and pBR328 (Prentki et al., 1981) . In pBR329 the Cmr gene lacks its original promoter but is transcribed counterclockwise toward the Apr gene by a promoter located to the right of the HindIII site in the Tcr gene.

Gene, 1982 Jan, 17(1), 65 - 73
Site-specific recA-independent recombination (fusion) of pMB8-related replicons in Escherichia coli in the region of the replication origins; Tomilin NV et al.; Escherichia coli recA+ and recA- cells were co-transformed with a mixture of pMB9 (Tcr) and pST8-26 (Apr, pBR325 derivative) plasmid DNAs followed by selection on plates containing both tetracycline and ampicillin . A set of stable Tcr Apr derivatives was isolated from these transformants . Many of the stable Tcr Apr segregants contained fused pMB9::pST8-26 plasmids with lengths that were about 0.8 kb longer than the sum of the lengths of the parental plasmids; one plasmid (pTF8) was about 1.5 kb shorter . The fusion was not stimulated by UV irradiation of co-transformants and occurred both in recA+ and recA- genetic backgrounds . Restriction analysis of the fused plasmids showed the two replicons were in the same relative orientation, and also indicated unique points of fusion in most cases (in 9 out of 10) which are localised within the 1.6-kb regions around the replication origins (RO) . Because the fusion of plasmids of the type used in this study was not described before we have tentatively named it RO-fusion (Replication-Origin-fusion).

Ann Microbiol (Paris), 1982 Jan, 133A(1), 77 - 80
Role of the catabolite activator protein in the expression of the maltose regulon of Escherichia coli; Chapon C; Using malT-lacZ strains deleted for gene crp, we have shown that the expression of malT is controlled by the catabolite activator protein (CAP), the product of gene crp . malT X mutations were obtained which allowed a malT-lacZ hybrid gene to be expressed at a high level even in the absence of CAP . These mutations were shown to be located in or close to the promoter of the malT gene . We transferred the malT X mutation cis to a wild type malT gene . In the resulting strains, the study of the expression of the three operons in absence or presence of CAP led us to the following conclusion . CAP appears to control malPQ expression mainly if not only by regulating the concentration of MalT protein in the cell . On the other hand it controls the two other operons more stringently both by regulating malT expression and by a more direct action probably exerted on the promoters of these operons.

Mol Biol (Mosk), 1982 Jan-Feb, 16(1), 55 - 8
{Molecular mechanisms of stabilization and preparation of metabolic breaks in DNA strands in vivo . I . Tandem action of exonuclease V and DNA polymerase II}; Fradkin GE et al.; Investigation of the formation of metabolic imbalance breaks in the DNA of thy- cells of E . coli during thymine starvation is described . The results of experiments indicate that two enzymes--exonuclease V and 3' exonuclease activity of DNA polymerase II take part in the formation of metabolic imbalance gaps . The manifestation of activity of the enzymes has a tandem character.

Cell, 1982 Jan, 28(1), 155 - 63
A symmetrical six-base-pair target site sequence determines Tn10 insertion specificity; Halling SM et al.; Transposon Tn10 inserts at many sites in the bacterial chromosome, but preferentially inserts at particular hotspots . We believe we have identified the target DNA signal responsible for this specificity . We have determined the DNA sequences of 11 Tn10 insertion sites and identified a particular 6 base pair (bp) symmetrical consensus sequence (GCTNAGC) common to those sites . The sequences at some sites differ from the consensus sequence but only in limited and well defined ways . The sequences at some sites differ from the consensus sequence than do sequences at other sites, and the consensus sequence and closely related sequences are generally absent from potential target regions where Tn10 is known not to insert . Other aspects of the target DNA can significantly influence the efficiency with which a particular target site sequence is used . The 6 bp consensus sequence is symmetrically located within the 9 bp target DNA sequence that is cleaved and duplicated during Tn10 insertion . This juxtaposition of recognition and cleavage sites plus the symmetry of the perfect consensus sequence suggest that the target DNA may be both recognized and cleaved by the symmetrically disposed subunits of a single protein, as suggested for type II restriction endonucleases . There is plausible homology between the consensus sequence and the very ends of Tn10, compatible with recognition of transposon ends and target DNA by the same protein . The sequences of actual insertion sites deviate from the perfect consensus sequence in a way which suggests that the 6 bp specificity determinant may be recognized through protein-DNA contacts along the major groove of the DNA double helix.

Proc Natl Acad Sci U S A, 1982 Jan, 79(1), 46 - 50
Transposon-specified site-specific recombination; Kitts P et al.; Cointegrate DNA molecules containing two copies of a transposable element appear to be intermediates in the transposition process . These structures are resolved by site-specific recombination to yield the normal end products of transposition . The transposable element gamma delta (Tn1000) synthesizes a product interchangeable with the Tn1/3tnpR protein in promoting Tn1/3 site-specific recombination . These data support the hypothesis that cointegrates containing directly repeated copies of Tn1/3 are obligatory intermediates in interreplicon transposition of Tn1/3 . In addition, we show here that the reaction is independent of the element-encoded tnpA gene product . Tn501, which specifies mercury resistance, also produces cointegrates as intermediates in interreplicon transposition . The appearance of Tn501-specified recombination activity that can act on these cointegrates requires growth of cells in the presence of Hg2+.

J Bacteriol, 1982 Jan, 149(1), 264 - 75
Mapping of a plasmid, coding for colonization, factor antigen I and heat-stable enterotoxin production, isolated from an enterotoxigenic strain of Escherichia coli; Smith HR et al.; The non-autotransferring plasmid NTP113 codes for production of colonization factor antigen I and heat-stable enterotoxin, NTP113, which has a molecular weight of 58 X 10(6), was digested with BamHI, EcoRI, and HindIII and combinations of these restriction endonucleases, and the products of these digestions were analyzed by agarose gel electrophoresis . The results were used to construct a partial restriction map of NTP113 . Transposons coding for resistance to ampicillin, kanamycin, and tetracycline were inserted into NTP113, and we obtained a series of deletion mutants, as determined by the loss of tetracycline or kanamycin resistance from strains carrying the insertion mutants . A number of plasmid mutants obtained by insertion or deletion did not code for colonization factor antigen I, but most of these mutants still coded for heat-stable enterotoxin production . The position of the inserted transposons and of the deletions were determined on the restriction map . Two regions of NTP113 were required for the expression of colonization factor antigen I, and the two sites were separated by a length of DNA corresponding to a molecular weight of about 25 X10(6).

J Bacteriol, 1982 Jan, 149(1), 191 - 7
Effect of catabolite repression on the mer operon; Summers AO et al.; The plasmid-determined mer operon, which provides resistance to inorganic mercury compounds, was subject to a 2.5-fold decrease in expression when glucose was administered at the same time as the inducer HgCl2 . This glucose-mediated transient repression of the operon was overcome by the addition of cyclic AMP . Permanent catabolite repression of the operon was observed in the 1.6- to 1.9-fold decrease in expression in mutants lacking either adenyl cyclase (cya) or the catabolite activator protein (crp) . The effect of the cya mutation on mer expression could be overcome by the addition of cyclic AMP at the time of induction, In addition to these effects on the whole cells of a wild-type strains, we examined the effect of catabolite repression on the expression of the mercuric ion {Hg(II)} reductase enzyme, assayable in cell extracts, and on the Hg(II) uptake system, assayable in a mutant strain which lacked reductase activity . There was a two- to threefold effect of repression on the Hg(II) reductase enzyme assayable in vitro after induction under catabolite repressing conditions (either with glucose or in the crp and cya mutants) . We did not find a similar repressing effect on the induction of the Hg(II) uptake system, which is also determined by the mer operon.

J Bacteriol, 1982 Jan, 149(1), 106 - 13
Insertion of transposons through the major cotransduction gap of Escherichia coli K-12; Fouts KE et al.; The major cotransduction gap of the Escherichia coli chromosome extends from mini 31 to 34 . We have inserted transposons through this gap which, by sequential transduction, link sbcA (min 29.8) with manA (min 35.7) and thus eliminate the gap . These results indicate that the length of DNA in the region, as measured by transduction, is not significantly different from the length obtained by conjugational time of entry . Since this segment of the E . coli chromosome has few known genes, these transposon insertions will be useful for genetic manipulations in the region of the gap . We describe the usefulness of these markers for rapidly mapping mutations which may be isolated in the region from min 27 to 37.

Arch Intern Med, 1982 Jan, 142(1), 133 - 4
Polymicrobial septicemia associated with rhabdomyolysis, myoglobinuria, and acute renal failure; Kalish SB et al.; Myoglobinuria and renal failure resulting from bacterial infection have only rarely been reported . To our knowledge, we describe the first reported case of polymicrobial septicemia resulting in rhabdomyolysis and myoglobinuric renal failure . Renal failure secondary to myoglobinuria has an excellent prognosis; in our patient, recovery was complete . The frequency of rhabdomyolysis, myoglobinuria, and renal failure in septicemia is unknown and can only be determined by an increased awareness of this potential complication of septicemia.

EMBO J, 1982, 1(8), 907 - 11
Phosphotransferase-mediated regulation of carbohydrate utilisation in Escherichia coli K12: identification of the products of genes on the specialised transducing phages lambda iex (crr) and lambda gsr (tgs); Britton P et al.; The expression of genes adjacent to ptsI was investigated using a series of specialised transducing phages carrying different, overlapping, segments of the cysA-gsr-ptsI-ptsH- iex - cysZ -lig region of the genome of Escherichia coli . The polypeptides were synthesised following the infection of u.v.-irradiated lysogenic and non-lysogenic uvrA recA hosts or a uvrA recA host carrying the lambda cI+ plasmid pKB280 . The polypeptides were identified by SDS-polyacrylamide gel electrophoresis and fluorography . The gsr gene product had a mol . wt . of 23 000 . The product of the iex gene was tentatively identified as a protein of mol . wt . of either 33 000 or 21 000 . Hpr, the product of the gene ptsH, had a mol . wt . of 9000 . The gsr gene appeared to be expressed at a higher level in a non-immune host, which suggests that it was transcribed from lambda promoters . A new lambda host strain, suitable for the detection of small polypeptides (mol . wt . less than 30 000) is described.

EMBO J, 1982, 1(5), 591 - 5
Homologies between different procaryotic DNA-binding regulatory proteins and between their sites of action; Gicquel-Sanzey B et al.; Comparison of the amino acid sequences of 13 procaryotic regulatory proteins, including the products of genes crp (catabolite activator protein; CAP), lacI, galR , lexA, lysR, araC, trpR, and tnpR of Escherichia coli, of genes cI, cII and cro of phage lambda, cro of phage 434, and c2 of phage P22, has revealed two regions of homology . The sites of action of these proteins also share common features in their DNA sequence . Taking into account the models proposed for the lambda repressors, cro and cI, and for CAP, a general type of DNA-protein interaction is suggested.

EMBO J, 1982, 1(1), 115 - 20
Z* DNA, the left-handed helical form of poly{d(G-C)} in MgCl2-ethanol, is biologically active; van de Sande JH et al.; The interconversion between the right (R) and left (L) helical forms of poly{d(G-C)} occurs at low concentrations of MgCl2 and EtOH, acting together in a highly synergistic manner . Thus, the cooperative R---L transition is induced by only 0.4 mM and 4 MM MgCl2 in combination with 20% and 10% EtOH, respectively . The L form of poly{d(G-C)} formed under these conditions has the spectroscopic properties (absorption, circular dichroism) previously demonstrated under high salt conditions (Pohl and Jovin, 1972) and thought to correspond to the left-handed Z DNA structures recently established by X-ray crystallography (Wang et al., 1979; Drew et al., 1980) . However, L DNA formed in Mg2+-EtOH (which we designate as Z* DNA) has unique properties: a) it can be sedimented readily out of solution at low speed, indicative of condensation and intermolecular aggregation; b) it supports the binding of several intercalating (ethidium bromide, actinomycin D) and non-intercalating (mithramycin) drugs, although these interact preferentially with the R (i.e., B) form of DNA; and c) it functions as a template for Escherichia coli RNA polymerase . B and Z* DNAs can be generated under identical ionic conditions and compared in a number of biochemical systems . Our results suggest that left-handed DNA may form under physiological conditions and serve a biological function.

Tokai J Exp Clin Med, 1982, 7 Suppl, 61 - 4
Na+-coupled transport of melibiose in Escherichia coli: analysis of mutants with altered cation specificity; Tsuchiya T et al.; Melibiose transport via the melibiose transport system of Escherichia coil utilizes either H+ or Na+ as a coupling cation . This system may represent a evolutional transition state between H+-substrate cotransport (bacteria type) and Na+-substrate cotransport (animal type) . We have isolated mutants which showed altered cation specificity for melibiose transport . The melibiose carrier of the mutants has lost the ability to recognize H+ . This communication describes the properties of the melibiose transport system and the properties of the mutants.

Microbios, 1982, 35(141-142), 169 - 78
Isoniazid perturbation of the pyridine nucleotide cycle of Escherichia coli; Heard JT Jr et al.; Isogenic strains of Escherichia coli, differing only in their sensitivity/resistance to isoniazid, were compared on the basis of intracellular accumulation of the intermediates of the pyridine nucleotide cycle and a closely related compound, nicotinamide adenine dinucleotide phosphate . Isoniazid treatment was bacteriostatic for the wild type organism and resulted in an exaggerated lag phase followed by a rapid logarithmic growth rate in the isoniazid-resistant strain . Isoniazid treatment of the wild type strain, even in the absence of growth, resulted in a temporal shift of peak pyridine accumulation from early lag phase to early logarithmic phase . The effect of mutation from isoniazid sensitivity to isoniazid resistance was accompanied by this same temporal shift of peak pyridine accumulation . In addition, mutation was accompanied by increases in the peak concentrations, as compared to wild type, of desamidonicotinamide adenine dinucleotide, nicotinamide, nicotinic acid and nicotinamide mononucleotide; the levels of nicotinic acid mononucleotide, nicotinamide adenine dinucleotide phosphate and nicotinamide adenine dinucleotide were decreased . Isoniazid treatment of the isoniazid-resistant strain, even in the absence of bacteriostasis, caused a decrease in the intracellular concentrations of nicotinic acid and nicotinamide and an increase in nicotinic acid mononucleotide . Isoniazid treatment of both the sensitive and resistant strains resulted in the loss of a peak ratio of NADP to NAD which normally occurred in the late logarithmic/early stationary phase of growth in untreated cultures . This denoted a loss of ability to shift from the generation of energy from glycolysis to generation of energy from the hexose monophosphate shunt.

Mol Gen Genet, 1982, 188(3), 459 - 64
A new activity in the Ftra operon which is required for F-pilin synthesis; Moore D et al.; Membrane preparations from a series of Hfr mutant strains of Escherichia coli K12 deleted in the promoter distal end of the F transfer operon were analyzed . Deletions which extended into traG, as expected, had no discernible effect on synthesis of membrane F-pilin . A more extensive deletion in strain K1777 which eliminated traH activity similarly had no effect on F-pilin synthesis . Membranes from three other TraF+ TraH- deletion strains, as well as membranes from all strains carrying deletions extending into traF or further, lacked F-pilin, however . Since traH amber mutations do not affect synthesis of membrane pilin (Moore et al . 1981 b) we conclude that a gene required for F-pilin biosynthesis is located between traF and traH . We have named this gene traQ . Further evidence for traQ and an assay for its activity was obtained by examining the products of a TraM+ TraJ+ TraA+ lambda transducing phage, KI lambda 13, in UV irradiated cells . Infection of F- cells with KI lambda 13 does not result in F-pilin synthesis . Membrane pilin is synthesized as a product of the transducing phage if an Flac or Hfr irradiated host is used, however . Mutant analysis demonstrated that this synthesis is independent of host expression of traA, traL, traE, traK, traB, traV, traW, traC, traU, traF, or traH, but dependent on expression of the traF-traH region . We interpret our data to indicate that an activity encoded by traQ is required for the conversion of traA product to F-pilin.

Mol Gen Genet, 1982, 187(1), 67 - 71
Suppressor genes of a dnaA temperature sensitive mutation in Escherichia coli; Takeda Y et al.; Recombinant plasmids were constructed from EcoRI digests of Escherichia coli chromosomal DNA and pMB9 DNA by selecting for suppression of a dnaA-T46 temperature-sensitive mutation . Two types of plasmid capable of suppressing the dnaA mutation were isolated . They did not carry any genetic markers around dnaA and physical mapping with various restriction enzymes showed that neither of the plasmids contained the dnaA gene . One plasmid, pYT47, was characterized further and the protein responsible for the suppression was identified by two-dimensional gel electrophoresis . The molecular weight of the suppressor protein was about 68 Kdal and thus is clearly different from the dnaA gene product.

Mol Gen Genet, 1982, 187(1), 30 - 6
Cloning of colicin E1 tolerant tolC (mtcB) gene of Escherichia coli K12 and identification of its gene product; Otsuji N et al.; A mutation in the tolC(mtcB) gene of Escherichia coli K12 results in increased sensitivity to sodium dodecylsulfate (SDS), sodium deoxycholate, basic dyes, mitomycin C, and bleomycin, and makes the cell tolerant to the killing action of colicin E1 . From lysogens with lambda cI857S7 integrated at a secondary attachment site, a transducing phage (lambda dtolC+) that transduces a tolC recipient to SDS resistance was isolated . A recombinant DNA molecule was constructed in vitro from plasmid pBR322 as a vector, and an EcoRI-BamHI fragment of lambda tolC+ DNA . The resulting plasmid, designated pOK1, was 5.6 megadaltons (Md) . The tolC bacteria transformed with plasmid pOK1 restored the TolC+ phenotype with regard to mitomycin C, SDS, and colicin E1 sensitivities . A plasmid with an amber mutation in the tolC gene, designated pOK18, was isolated by the same procedure used for the isolation of pOK1 . The plasmid had a molecular weight of 5.6 Md and produced the same size of DNA fragments as the tolC+ plasmid, pOK1, after digestion with the indicated restriction enzymes . The plasmid, pOK18, conferred the TolC+ phenotype when introduced into a tolC strain in the presence of, but not the absence of, an amber suppressor . Plasmid-specified polypeptides were determined by using maxicells of strains uvrA recAsup+ and uvrA recA tyrT, containing each plasmid . Three additional proteins of 54,000 (54K), 29K, and 27K were produced in maxicells containing pOK1 . These three proteins were synthesized in maxicells of the uvrA recA tyrT strain carrying pOK18, whereas synthesis of the 54K protein by pOK18 did not take place in maxicells of the uvrA recA sup+ strain, although the other two proteins were produced in normal amounts . From these results we concluded that the product of the tolC gene is a protein with a molecular weight of 54K.

Mol Gen Genet, 1982, 188(1), 37 - 43
Cell survival, UV-reactivation and induction of prophage lambda in Escherichia coli K12 overproducing RecA protein; Quillardet P et al.; The effect of the cellular level of RecA protein on the ability of E . coli K12 bacteria to (i) survive UV-irradiation (ii) promote UV-reactivation of UV-damaged phage lambda (iii) induce prophage lambda was determined in bacterial mutants with discrete increasing levels of RecA protein . The various levels of RecA protein were obtained by combining lexA and recA alleles . Except for the double mutant lexA3 recAo98, whose repair ability was 25% less than that observed in wild type bacteria, bacterial survival was proportional to the level of RecA protein measured after 90 min of incubation . In lexA3 recAo98 bacteria, RecA protein, at a constitutive high basal level, failed to compensate totally for the lack of LexA repressor cleavage; UV-reactivation of UV-damaged phage lambda was not restored; yet, prophage lambda was induced with 35% efficiency . Efficient UV-induction of prophage lambda is linked to the induction of lexA-controlled host processes that repair the UV-damaged prophage.

Mol Gen Genet, 1982, 187(3), 459 - 60
Trans-dominant mutation affecting restriction and modification in Escherichia coli K-12; Hubacek J et al.; We have analysed the mechanism of action of a ts mutation in E . coli, which has an effect on the expression of the restriction and modification phenotype . The frequencies of recombinants obtained in transduction experiments support the idea that the temperature sensitive mutation is located outside the hsd operon in the gene denoted hsd.X . Complementation experiments demonstrated the trans-dominant nature of the temperature sensitive mutation . The possible role of the hsd.X product in the formation of EcoR.K and EcoM.K complexes and their interaction with the recognition site on the DNA is discussed.

Mol Gen Genet, 1982, 187(2), 330 - 4
Temperature-sensitive mutant rho-115 rho-RNA binary complexes, and stabilization by substrates and analogues; Kent RB et al.; To determine the molecular basis for the temperature-sensitivity of pure rho RNA-dependent ATPase from Escherichia coli mutant rho-115 cells, we investigated mutant rho binding to {3H} polyC as measured by retention on nitrocellulose filters . Complexes of wild-type rho and polyC incubated at 37 degrees C and 45 degrees C were similarly stable . At 37 degrees C mutant rho-polyC binary complexes were inactivated at a slightly faster rate than complexes with wild-type rho . Upon shift to 45 degrees C the quantity of rho-115 bound to polyC declined immediately, resulting in one-fifth of the quantity of complexes observed at 37 degrees C . Shift back to 37 degrees C restored the level of observed complexes by two-fold . The inclusion of ATP or the analogue beta-gamma methylene ATP during 45 degrees C incubation resulted in stable mutant rho-polyC complexes . The hydrolysis product ADP was also effective in stabilizing binary complexes at 45 degrees C but this effect was observed with an order of magnitude more ADP than ATP . Adenine, adenosine, AMP or Pi had no stabilizing effect . We conclude that the mutant rho-115 protein exhibits a structural instability as a result of binding RNA . Furthermore ATP confers a wild-type phenotype upon rho-115 protein, probably as a result of conformational change due to binding of this compound . The effect of ATP on the stability of mutant rho-polyC binary complexes supports the model of ATP modulation of rho-RNA interaction proposed by Galluppi and Richardson (1980).

Mol Gen Genet, 1982, 186(3), 333 - 8
Role of SSB protein in RecA promoted branch migration reactions; West SC et al.; The RecA protein of Escherichia coli is essential for genetic recombination and postreplicational repair of DNA . In vitro, RecA protein promotes strand transfer reactions between full length linear duplex and single stranded circular DNA of phi X174 to form heteroduplex replicative form II-like structures (Cox and Lehman 1981 a0 . In a similar way, it transfers one strand of a short duplex restriction fragment to a single stranded circle . Both reactions require RecA and single strand binding protein (SSB) in amounts sufficient to saturate the ssDNA . The rate and extent of strand transfer is enhanced considerably when SSB is added after preincubation of the DNA with RecA protein . In contrast, SSB protein is not required for RecA protein catalysed reciprocal strand exchanges between regions of duplex DNA . These results indicate that while SSB is necessary for efficient transfer between linear duplex and ssDNA to form a single heteroduplex, it is not required for branch migration reactions between duplex molecules that form two heteroduplexes.

Basic Life Sci, 1982, 20, 179 - 97
In vitro replication of mutagen-damaged DNA: sites of termination; Moore PD et al.; We have examined the effect of DNA lesions, which in vivo are potentially mutagenic, on in vitro DNA synthesis carried out by a number of purified DNA polymerases using a 0X174 template . Both acetyl aminofluorene (AAF) adducts and UV-induced pyrimidine dimers are blocks to elongation by DNA polymerases . On UV-irradiated DNA templates synthesis terminates one nucleotide before the sites of pyrimidine dimers with all of the enzymes tested: Pol I and Pol III holoenzyme from Escherichia coli, T4 DNA polymerase, avian myeloblastosis virus reverse transcriptase and a mammalian DNA polymerase alpha . With AAF, which reacts at the C-8 position of guanine, differences are observed between the above enzymes, with the latter two inserting a nucleotide opposite the site of the lesion . Substitution of Mn2+ for Mg2+ as the cation in the Pol I reactions causes changes in the termination pattern on both UV-irradiated and AAF-reacted templates . The significance of these results to the process of inducible error-prone repair and the possible bypass of lesions in the DNA is discussed.

Mol Gen Genet, 1982, 186(2), 170 - 9
Cleavage of lambda repressor and synthesis of RecA protein induced by transferred UV-damaged F sex factor; Moreau PL et al.; Transfer of a UV-damaged F sex factor to a recipient lambda lysogen induces prophage lambda development . Under these conditions RecA protein synthesis was induced and lambda repressor cleaved, as observed upon direct induction, that is, when the recipient lambda lysogen was directly exposed to UV-light . The efficiency of induction of RecA protein synthesis in recipient bacteria which had received an irradiated F-lac factor was about 80% of that measured upon direct induction . We observed the simultaneous disappearance of lambda repressor and a slight production of cleavage fragments; quantitation by densitometric scanning of the autoradiogram after correction for the efficiency of transfer indicated that 55% of lambda repressor was cleaved . Transfer of UV-damaged Hfr DNA failed to induce RecA protein synthesis . A lambda phage vector carrying oriF, the cloned origin of F plasmid replication, after exposure to UV-light and infection of a recipient lysogen, induced RecA protein synthesis and a moderate but significant cleavage of lambda repressor . Indirect induction by UV-damaged F sex factor or phage lambda oriF resulted in biochemical cellular reactions similar to those observed upon direct induction . LexA repressor that negatively controls RecA protein synthesis appeared more susceptible to cleavage than did lambda repressor.

Mol Gen Genet, 1982, 186(1), 111 - 7
UV-induced allevation of lambda restriction in Escherichia coli K-12: kinetics of induction and specificity of this SOS function; Thoms B et al.; In UV-irradiated cells of Escherichia coli K-12 a partial release of the restriction of non-modified phage lambda is observed when the cells are recA+ lexA+ . We show here that the induction of this restriction allevation (RA) also depends on the recBC enzyme and that the expression of RA requires protein synthesis . Maximum expression was reached within 60 to 90 min after irradiation . Experiments are presented which show that upon UV-irradiation a signal is created which triggers the development of RA when protein synthesis is allowed . This signal decayed with a half-life of only a few minutes in cells treated with chloramphenicol . The decay kinetics were similar in uvr+ and uvrA mutants . RA appeared to be specific for EcoK insofar as no allevation of lambda restriction by EcoRI, EcoRII and EcoP1 occurred . During maximum expression of RA no gross reduction of the activities of the recBC enzyme (exonuclease V) and the restriction endonuclease EcoK was observed and no new DNA modifying activity appeared in the cells . Since, in fully expressed cells, up to 75% of the infecting lambda DNA was converted to acid-soluble material within 20 min after infection we suggest that only a small specific fraction of lambda infections may undergo RA.

Mol Gen Genet, 1982, 185(3), 445 - 7
An amber mutation in the gene rpsA for ribosomal protein S1 in Escherichia coli; Kitakawa M et al.; An amber mutation has been induced in the gene rpsA (which codes for ribosomal protein S1) of Escherichia coli K-12 strain in the presence of an amber suppressor (supD) and mutations sueA, sueB and sueC that additively enhance the efficiency of suppression . That the amber mutation has occurred in the gene rpsA was confirmed by complementation with a plasmid which carried the wild-type allele of rpsA . The mutation is lethal in the absence of an amber suppressor, indicating that ribosomal protein S1 is indispensable to E . coli.

Mol Gen Genet, 1982, 185(3), 430 - 9
Quantitative evaluation of recA gene expression in Escherichia coli; Casaregola S et al.; A recA::lac operon fusion was constructed using the phage Mu d(Ap, lac) in Escherichia coli to obtain precise measurements of the level of recA gene expression in various genetic backgrounds . The RecA protein normally represents 0.02% of total protein . This value is known to increase dramatically after treatments interrupting DNA synthesis; kinetic experiments showed that the rate of recA expression increases 17-fold within 10 min after UV irradiation or thymine starvation . In mutants affected in SOS regulation or repair the following observations were made: (i) the tif-1 mutation in the recA gene does not alter the basal level of recA expression, suggesting that it improves the protease activity of RecA; (ii) the lexA3 mutation does not create a "super-repressor" of recA; (iii) the tsl-1 mutation in the lexA gene makes the LexA protein a poor repressor of recA at 30 degrees C (2.5-fold derepression) and a poor substrate for RecA protease (3-fold stimulation of recA expression by UV); (iv) the spr-55 amber mutation in the lexA gene causes a 30-fold increase in recA expression, higher than all inducing treatments, and this level cannot be further increased by nalidixic acid; (v) the zab-53 mutation at the recA locus, known to abolish tsl-mediated induction of recA expression, is trans-recessive and thus probably affects a regulatory site on the DNA; (vi) uvrA, B and C, recB and recF mutations do not increase the basal level of recA expression, suggesting that there are not sufficient spontaneous lesions to cause induction even when any one of these three repair pathways is inoperative.

Mol Gen Genet, 1982, 185(1), 43 - 50
Constitutive expression of SOS functions and modulation of mutagenesis resulting from resolution of genetic instability at or near the recA locus of Escherichia coli; Witkin EM et al.; Cellular activities normally inducible by DNA damage (SOS functions) are expressed, without DNA damage, in recA441 (formerly tif-1) mutants of Escherichia coli at 42 degrees C but not at 30 degrees C . We describe a strain (SC30) that expresses SOS functions (including mutator activity, prophage induction and copious synthesis of recA protein) constitutively at both temperatures . SC30 is one of four stable subclones (SC strains) derived from an unstable recombinant obtained in a conjugation between a recA441 K12 donor and a recA+ B/r-derived recipient . SC30 does not owe its SOS-constitutive phenotype to a mutation in the lexA gene (which codes the repressor of recA and other DNA damage-inducible genes), since it is lexA+ . Each of the SC strains expresses SOS functions in a distinctively anomalous way . We show that the genetic basis for the differences in SOS expression among the SC strains is located at or very near the recA locus . We propose that resolution of genetic instability in this region, in the original recombinant, has altered the pattern of expression of SOS functions in the SC strains.

EMBO J, 1982, 1(9), 1081 - 8
Simian virus 40 cRNA is processed into functional mRNA in microinjected monkey cells; Graessmann M et al.; Monkey cells, microinjected with simian virus 40 (SV40) in vitro synthesized cRNA produce full-size tumor (T)-antigen . This was verified by analyzing immunoprecipitates of microinjected cells by polyacrylamide gel electrophoresis . Early SV40 DNA contains an intron within the large T-antigen coding sequences . Therefore, cRNA copied in vitro from the early DNA strand requires removal of the intron in order to become a functional mRNA . Polyadenylation of the cRNA in vitro by Escherichia coli poly(A)-polymerase increased the biological activity of the RNA . Detection of T-antigen by gel electrophoresis required as little as 50 poly(A)-cRNA injected cells . Splicing of the microinjected cRNA appears to be a nuclear process . Cells enucleated by cytochalasin B prior to injection do not synthesize large T-antigen . However, small t-antigen, a protein with a continuous sequence, is synthesized in these cells . Finally, it is shown that the process of splicing is not required for the transport of mRNA from the nucleus into the cytoplasm . Authentic T-antigen mRNA, isolated from virus infected cells, induced T-antigen synthesis with similar efficiency after either nuclear or cytoplasmic injection.

Tokai J Exp Clin Med, 1982, 7 Suppl, 33 - 42
Perspectives for achieving improved information by the observation of thin sections in the electron microscope; Carlemalm E et al.; Obtaining biologically significant fine detailed information from sections is limited firstly by the unknown distribution of heavy metal stain and secondly by conformational changes induced by dehydration . Only afterwards we have to be concerned with beam induced alterations . By Z-imaging in a STEM, completely unstained material can now become imaged sharply with high contrast . By this the first limitation is eliminated and the second can become explored . For this purpose new resins designed for low temperature embedding might become important . First biological results are presented which illustrate the potential of these techniques: The transmembrane protein of the separate junction has been revealed as such and shown that only the hydrophilic part of the protein is stained by uranyl acetate . This type of staining therefore does not allow the detection of any transmembrane proteins in sections.

Princess Takamatsu Symp, 1982, 12, 1 - 22
Structure and expression of human alpha-interferon genes; Weismann C et al.; cDNA was prepared from induced leukocyte poly(A) RNA and cloned in Escherichia coli . Interferon (IFN)-alpha cDNA clones were isolated by subculture cloning using a translation hybridization assay . Definitive identification of the clones was based on the production of an interferon-like protein by the transformed bacteria . Different IFN-alpha cDNAs, with characteristic target cell specificities, were identified . The cloned cDNAs typically encode a mature polypeptide of 166 (or, in the case of IFN-alpha 2, 165) amino acids and a signal sequence of 23 amino acids.

Microbiol Immunol, 1982, 26(9), 779 - 93
R483 and F plasmid genes promoting RNA degradation: comparative restriction mapping; Akimoto S et al.; The gene promoting nucleic-acid degradation (pnd) on IncIa plasmid R483 was cloned into pBR322 . It is located on a 0.85 kilobase (kb) EcoRI-SalI fragment and is close to Tn7 . The pnd gene has similar properties to the srnB gene on the F plasmid . A cleavage map of the 0.85 kb pnd fragment was constructed and compared with that of the 1.18 kb EcoRI-BamHI fragment containing the srnB gene . These two regions showed marked heterogeneity as evidenced by their distinctly different restriction maps . This result suggests separate paths of evolution of the two genes for stable RNA degradation.

Can J Biochem, 1982, 60(10), 952 - 61
Novel aspects of the structure of the Escherichia coli nucleoid investigated by a rapid sedimentation assay; Lee JS et al.; The Escherichia coli nucleoid is maintained in its folded highly condensed state by constraints which involve RNA and protein . We have developed a rapid sedimentation assay to determine the state of folding of the membrane-free nucleoid . An approximate measure of the stability of the nucleoids under various conditions can then be estimated by measuring the temperature at which the nucleoids unfold . Using ethidium and gamma irradiation (which removes the negative supercoiling of the native nucleoid) as probes, it can be shown that there are two types of constraint involved in the condensation of the nucleoid . One of these constraints is destabilized by ethidium but stabilized by negative supercoiling; the second constraint is unaffected by both ethidium and negative supercoiling . Several models can be proposed: (i) a DNA . RNA duplex, (ii) a double-strand DNA (dsDNA) . RNA triplex, (iii) DNA-protein interactions, (iv) a topological knot with RNA, and (v) a DNA tetraplex . The topological knot model is not consistent with the data and many combinations of the others can be excluded . If RNA is involved in both constraints then RNA . DNA duplexes and dsDNA . RNA triplexes are involved in stabilizing the nucleoid.

Cytogenet Cell Genet, 1982, 34(3), 193 - 203
Construction of cloned libraries from RNA of human fetal tissues; Kurnit DM et al.; We have constructed libraries of recombinant DNA plasmids containing sequences complementary to polyadenylated RNA from a variety of human midtrimester fetal tissues . The bacterial colonies containing these plasmids have been grown and replicated on nitrocellulose filters in a manner that facilitates permanent storage, rapid screening, and transportability to other laboratories . We screened a portion of the library for the presence of repetitive sequences and found that approximately 20% of the clones contain repetitive sequences . We have also shown that some clones contain nonrepetitive sequences . Pools of recombinant cDNA-containing plasmids devoid of repetitive sequences have been constructed to permit the chromosomal localization of a variety of actively transcribed sequences . The construction of such large, tissue-specific clone banks should facilitate the direct isolation, mapping, and characterization of normal and abnormal human genes.

Oncodev Biol Med, 1982, 3(4), 301 - 13
Alphafetoprotein messenger RNA in human embryonal carcinoma grown in nude mice, and cloning of its complementary DNA; Morinaga T et al.; Messenger RNA for human apha-fetoprotein (AFP) was partially purified from human testicular embryonal carcinoma grown in nude mice . DNA complementary to the mRNA was synthesized, tailed with oligo(dC) and inserted at the PstI site of the plasmid pBR322 tailed with oligo(dG) . Escherichia coli strain LE392 was transformed with the chimeric plasmid and colonies were screened for human AFP mRNA sequences by hybridization with DNA complementary to mouse AFP mRNA (greater than 95% pure) . Five colonies showed positive hybridization . The plasmid pHAF2, containing the largest insert, 900 nucleotides, was further characterized by hybrid-arrest of translation of AFP mRNA, restriction endonuclease mapping and partial nucleotide sequence analysis . It was found that the AFP complementary DNA (cDNA) sequence in pHAF2 corresponded to the 3'-end of AFP messenger RNA (mRNA), and showed a restriction map entirely different from that of mouse AFP cDNA clones previously reported.

Dev Comp Immunol, 1982 Summer, 6(3), 491 - 8
Limitations in response capacity of the newt, Notophthalmus viridescens, to soluble and particulate antigens; Ruben LN et al.; We have investigated the cellular basis of the failure of primitive extant vertebrates, e.g . Notophthalmus viridescens, the American common newt, to respond to soluble thymus-dependent (TD) antigens, e.g . keyhole limpet hemocyanin (KLH) . We have found that KLH can be used to prime for amplification of a response to hapten, 2,4,6-trinitrophenyl (TNP) when it is conjugated to KLH, only if both the unconjugated and conjugated KLH are adsorbed onto bentonite particles . The response is monitored in terms of antigen binding cells in the spleen . Moreover, prior injection of colloidal carbon, which is actively engulfed by phagocytes throughout the body, will prevent the response from taking place . Injection of colloidal carbon diminished responses to heterologous erythrocytes and to soluble or bentonite-coated 2,4-dinitrophenylated (DNP) dextran . Comparable colloidal carbon treatment did not reduce response levels to either soluble or bentonite adsorbed TNP-lipopolysaccharide (LPS) of E . coli . We suggest that the limitation in response capacity to soluble TD antigens may be associated with regulatory phagocytes . Moreover, the phagocytic cells of this primitive vertebrate appear to mediate responses to naturally particulate TD antigens and certain thymus-independent (TI) carriers, e.g . dextran, but not to LPS.

Mol Gen Genet, 1982, 186(3), 378 - 84
Cloning and expression of the ilvB gene of Escherichia coli K-12; Newman T et al.; A plasmid containing the ilvB operon, which codes for acetohydroxy acid synthase I of Escherichia coli K-12, was isolated using a ligated mixture of DNA from plasmid pBR322 and FilvB4 treated with endonuclease SalI . A shortened derivative of this plasmid was isolated by cloning a 3.4 kb bacterial fragment into plasmid pKEN005 to yield plasmid pTCN12 . The orientation of the ilvB operon relative to plasmid genes was determined by restriction enzyme mapping . Measurement of the level of the product of the ilvB gene, acetohydroxy acid synthase I, indicated that plasmid pTCN12 contained a functional ilvB promoter and control region . The DNA frm this plasmid was used as a probe to show that the rate of synthesis of ilvB mRNA was proportional to the levels of acetohydroxy acid synthase I.

Mol Gen Genet, 1982, 186(2), 221 - 7
Nonsense suppression in aminoacyl-t-RNA limited cells; Gallant J et al.; A number of nonsense alleles of lacZ exhibit phenotypic suppression (as much as a sixteen-fold increase in leakiness) during partial limitation for certain aminoacyl-tRNA species in relA mutant cells . Each responsive allele has its individual pattern of response to limitation for one or more amino acids or aminoacyl-tRNA's . The phenotypic suppression occurs only during limitation, and ceases once limitation is reversed . Suppression is much reduced by the presence of the relA+ allele or an allele of rpsL which restricts ribosomal ambiguity . In one case, the suppressed product has been identified by radioimmune assay and gel electrophoresis, and is a full-length lacZ protomer . Mechanisms are discussed whereby aberrations of translation at codons calling for an aminoacyl-tRNA species in short supply might lead to readthrough of a nearby nonsense codon.

Microbiol Immunol, 1982, 26(1), 41 - 57
Intranuclear incorporation of thymic low molecular weight RNA by murine bone marrow immunoblasts and inhibition of plasma cell formation by a derivative of rifampicin; Nakamura K et al.; An in vitro culture system for the proliferation of IgG-forming plasma cells from mouse bone marrow cultures has previously been described . The present study attempts to elucidate the mode of action of thymic RNA in these cultures . Autoradiography after using radiolabeled thymic RNA showed that radioactive material was mainly incorporated into the nuclei of IgG-forming plasma cells . No radiolabeled thymic RNA was incorporated into the cells except immunoblasts . The incorporated thymic RNA was acid insoluble and digested by RNase, but resistant to DNase and pronase . Radioactivity in the nucleotide pool after the cells were cultured with radiolabeled thymic RNA was negligible, indicating that reutilization of degraded RNA did not occur in the nuclei of the plasma cells . Moreover, the incorporation of radiolabeled thymic RNA by the cells was not prevented by excess unlabeled nucleosides . Escherichia coli transfer RNA, L-cell RNA and synthetic polynucleotide poly(A-U) were incorporated but were distributed in a different manner in the cells . A derivative of rifampicin, 2'5'-dimethyl N(4') benzyl-N(4'){desmethyl}rifampicin (AF/ABDMP), a possible inhibitor of RNA-dependent DNA polymerases, suppressed both the incorporation of thymic RNA and the differentiation of immunoblasts . AF/ABDMP suppressed DNA synthesis by bone marrow cultures to the same level as those pretreated with anti-mouse B-cell antibodies and complement . DNA dependent RNA polymerase activity was observed in the supernatant of bone marrow cultures stimulated by normal syngeneic thymic RNA and human gammaglobulin as antigen . These results imply a possible relationship between B-cell differentiation and RNA-dependent DNA polymerases.

Biochem J, 1982 Jan 1, 201(1), 145 - 51
Mechanism of inhibition of Escherichia coli RNA polymerase by captan; Dillwith JW et al.; Captan (N-trichloromethylthiocyclohex-4-ene-1,2-dicarboximide) was shown to inhibit RNA synthesis in vitro catalysed by Escherichia coli RNA polymerase . Incorporation of {gamma-32P}ATP and {gamma-32P}GTP was inhibited by captan to the same extent as overall RNA synthesis . The ratio of {3H}UTP incorporation to that of {gamma-32P}ATP or of {gamma-32P}GTP in control and captan-treated samples indicated that initiation was inhibited, but the length of RNA chains being synthesized was not altered by captan treatment . Limited-substrate assays in which re-initiation of RNA chains did not occur also showed that captan had no effect on the elongation reaction . Studies which measured the interaction of RNA polymerase with template DNA revealed that the binding of enzyme to DNA was inhibited by captan . Glycerol-gradient sedimentation of the captan-treated RNA polymerase indicated that the inhibition of the enzyme was irreversible and did not result in dissociation of its subunits . These data are consistent with a mechanism in which RNA polymerase activity was irreversibly altered by captan, resulting in an inability of the enzyme to bind to the template . This interaction was probably at the DNA-binding site on the polymerase and did not involve reaction of captan with the DNA template.

Mol Biol (Mosk), 1982 Jan-Feb, 16(1), 47 - 54
{Synthesis and cloning of DNA, complementary to rabbit globin pre-mRNA}; Zolotukhin SB et al.; cDNA synthesized on rabbit bone marrow erythroid cells pre-mRNA was cloned in bacterial plasmids . Cold phenol extracted pre-mRNA was a several times more effective template in the reaction of reverse transcription without oligo(dT) 10-primer than hot phenol extracted pre-mRNA . There was no yield increase of DNA-product on hot phenol extracted pre-mRNA in the reaction of reverse transcription with the oligo(dT)10-primer addition . The "hot phenol" poly (A)+-pre-mRNA was used to obtain the representative, full-sized cDNA . The double-stranded form of this cDNA, obtained with the help of DNA-polymerase I, was inserted into the PstI-site of pBR322 plasmid . About 25% E . coli JC5183 clones, transformed with this hybrid plasmid, were found to contain the globin sequences.

Mol Biol (Mosk), 1982 Jan-Feb, 16(1), 217 - 23
{Interactions of non-histone proteins with immobilized components of chromatin}; Fainboim AM et al.; The specific interaction between non-histone proteins (NHP) of rat thymus and the dextran-immobilized components of chromatin has been investigated . DNA's from E . coli and rat thymus, total histone and reconstituted nucleohistone were used as the affinity sorbents . The distribution of protein fractions in the NHP groups dissociated from chromatin with different ionic strengths was studied by binding on the columns and by SDS-PAAG-electrophoresis . It is shown that NHP of chromatin include the protein components with selective affinity to nucleohistone . These results are discussed in connection with the different functions of NHP in chromatin.

Eur J Biochem, 1982 Jan, 121(2), 383 - 9
The composition and properties of the Escherichia coli 5-S RNA-protein complex; Metspalu E et al.; At a high concentration of MgCl2 (30 mM) and a low concentration of proteins from the 50-S subunit (0.2 mg/ml), only three proteins, L15, L18 and L25, bind to 5-S RNA in significant amounts . On the other hand, in a buffer containing only 1 mM Mg Cl2, but otherwise at the same ionic strength (0.2 M), or at a protein concentration about 1.5 mg/ml, a large, stable complex can form between immobilized 5-S RNA and 50-S ribosomal proteins . This complex contains proteins L2, L3, L5, L15, L16, L17, L18, L21, L22, L25, L33 and L34, and it possess properties relevant to the function of the 50-S subunit; it has a binding site for deacylated tRNA, with a dissociation constant of 4.5 x 10(-7) M . The complex formed with 5-S RNA immobilized on an affinity column interacts also with 30-S subunits . The 5-S RNA-protein complex is interpreted as a sub-ribosomal domain which includes a considerable fraction of the peptidyl transferase center of the Escherichia coli ribosome.

EMBO J, 1982, 1(3), 379 - 84
Nucleotide sequence of the asd gene of Escherichia coli: absence of a typical attenuation signal; Haziza C et al.; The asd gene of escherichia coli encodes aspartic semialdehyde dehydrogenase, an enzyme involved in lysine, threonine, and methionine biosynthesis; its synthesis is controlled by a multivalent repression mechanism . It was cloned in plasmid pBR322 and its complete nucleotide sequence determined . The sequence predicts a polypeptide chain of 367 amino acids, in good agreement with results obtained for the purified protein ( Biellmann et al., 1980a ) . Our data indicate a Cys residue instead of a His residue, which was proposed after covalent labeling of the active center of the enzyme; this is more in line with the catalytic site of glyceraldehyde-3-phosphate dehydrogenase, an enzyme which carries out a similar reaction . The nucleotide sequence that precedes the translational start does not display any of the characteristic features of an attenuation signal . Hence the expression of the asd gene is probably not controlled in the same way as other multivalently repressed operons such as ilva and thr.

Mol Gen Genet, 1982, 187(2), 316 - 9
Fusion of the lac genes to the promoter for the aminopeptidase N gene of Escherichia coli; Murgier M et al.; Strains of Escherichia coli K12 were isolated in which the lac structural genes were fused to the promoter for the aminopeptidasee N structural gene (pepN) . Although this enzyme is constitutively produced, the differential rate of synthesis is increased about 4-fold upon phosphate starvation . The pepN-lac fusions were shown to respond to phosphate specific regulatory signals . A plaque forming lambda transducing phage bearing the pepN-lac fusion was isolated . This phage was used to prove genetically the fusion of lac genes to the promoter for the aminopeptidase . These results demonstrate a control at the transcriptional level of aminopeptidase synthesis.

Med Microbiol Immunol (Berl), 1982, 171(2), 85 - 90
The occurrence of colonisation factors (CFA/I, CFA/II and E8775) in enterotoxigenic Escherichia coli from various countries in South East Asia; Thomas LV et al.; Four hundred and fifty-eight enterotoxigenic strains of Escherichia coli (ETEC) were examined for the presence of colonisation factor antigens (CFA) I and II, and the putative colonisation factor, E8775, using an immunodiffusion technique with specific antisera . The ETEC strains had been isolated in Thailand, Bangladesh and from travellers returning to Japan from abroad . Approximately 14% of the ETEC strains possessed CFA/I and a further 13% of the strains possessed CFA/II . The E8775 antigen was found on 5% of the strains . CFA/I was found on strains of the serogroups 04, 015, 063, 078, 090, 0110, 0126, 0128, 0153, and 0? CFA/II was found on strains of the serogroups 06, 08, 09, 078, 0115, 0139, 0? and 0 rough . The E8775 antigen was found on strains of the serogroups 025, 0115, and 0167 . The results of this study emphasise the need to continue the search for other mechanisms of adhesion used by ETEC strains, and in particular strains of the serogroups 027, 034, 0148 and 0166.

Acta Microbiol Acad Sci Hung, 1982, 29(2), 129 - 35
Pilus antigen 987P produced by strains of Escherichia coli serotypes O141:K--, H- and O8:K85:H--; Nagy B et al.; Escherichia coli strains isolated from the alimentary tract of 68 weaned and 44 unweaned pigs with diarrhoea in various parts of Hungary, were tested for the presence of pilus antigens K88, K99 and 987P . K88 was detected in 30% of the strains from newborn pigs and in 12% of the strains isolated from weaned pigs . One strain carried K99 . Based on agglutination test and immunoelectron microscopic studies with specific absorbed antisera, five non-haemolytic E . coli strains isolated from newborn pigs were found to produce so-called 987P pili . Three of these strains were designated serologically as O8:K85:H--,987P+ and two as O141: K--:H--,987P+ . The Y1 cell assay, the infant mouse assay, and the ligated intestinal loop assay in less than 3-week-old pigs indicated that none of the strains produced heat-labile enterotoxin but all produced a heat-stable enterotoxin detectable in infant mice and in pig loops (STa) . All the strains induced diarrhoea in newborn, colostrum deprived pigs and colonized the lower small intestine by adhesion to the villous epithelium . The results have confirmed earlier findings about adhesive virulence attributes caused by 987P pili.

Scand J Infect Dis Suppl, 1982, 33, 83 - 95
Reactogenicity, immunogenicity and efficacy studies of Escherichia coli type 1 somatic pili parenteral vaccine in man; Levine MM et al.; Purified type 1 somatic pili from enterotoxigenic Escherichia coli (ETEC) strain H10407 (O78:H11) was evaluated as a parenteral immunizing agent in the hope that this antigen might enhance a contemplated polyvalent pilus vaccine . Intramuscular inoculation with 45, 90, 900 or 1 800 mcg of pili vaccine was tolerated without incident in 82 volunteers . Six of 15 persons who received a 28 day booster of 1 800 mcg developed local reactions while none of 52 persons receiving 180 or 450 mcg boosters evinced such reactions . Pili vaccine did not significantly alter intestinal transit time, absorptive capacity or the prevalence of colonic E . coli bearing type 1 somatic pili of the H10407 antigenic variety . All vaccinees developed significant rises in circulating IgG antibody to type 1 somatic pili, the magnitude of the response being directly proportioned to the vaccine dose . None of the vaccinees had significant rises to CFA I or II pili nor to heat-labile enterotoxin . However, many had rises in O antibody, particularly among those inoculated with 1 800 mcg . Three challenge studies were carried out with E . coli H10407 to assess vaccine efficacy . In the initial study the vaccinees were either protected against diarrhea (2 of 6 vaccinees versus 7/7 of controls) or had milder disease than the controls . In two subsequent challenges with H10407 significant protection was not seen . It was not clear whether protection exhibited by the vaccinee group in the first challenge was due to O antibody, pili antibody, or both acting synergistically . To clarify this, a group of the immunized volunteers were challenged with ETEC strain B7A which is a different serotype (O148:H28) lacks CFA/I or II pili, but possesses type 1 somatic pili antigenically distantly related to those of H10407 . Attack rates and severity of illness were similar in both vaccinee and control groups . While most volunteers challenged with E . coli H10407 developed significant rises in circulating antibody to CFA/I, LT and O antigen, none had rises to type 1 somatic pili . It is unclear if this is due to immune tolerance to this antigen when encountered enterally or whether these pili are not present in vivo in ETEC initiating diarrhea in the proximal small intestine . In summary, parenterally inoculated type 1 somatic pili were safe and highly immunogenic in man but did not consistently induce protection . Further studies are planned to clarify the role of antibody to type 1 somatic pili in mediating protection.

Mol Gen Genet, 1982, 186(3), 405 - 10
Regulation of expression of the dadA gene encoding D-amino acid dehydrogenase in Escherichia coli: analysis of dadA-lac fusions and direction of dadA transcription; Wild J et al.; The catabolism of most d-amino acids is carried out by d-amino dehydrogenase, coded by the dadA gene . Employing Mud(Aprlac) phage (Casadaban and Cohen 1979) the lac structural gene were fused to the control region of the dadA locus . Expression the of the lacZ gene in the dad-lac fusions was shown to be inducible by alanine and catabolite-repressible, thus responding to regulatory signals known to affect expression of the dadA gene . The method of MacNeil et al . (1980) was adopted to transfer the chromosomal dadA-lac fusion into lambda phage . By use of isolated lambda ddadA-lac1 phage, the promoter for the dadA gene was located, which enabled determination of the direction of dadA transcription as being counter-clockwise.

Mol Gen Genet, 1982, 186(2), 282 - 8
Use of a known plasmid and transposon as tracers for the incompatibility classification of a cryptic plasmid; Judge MS et al.; We have developed a novel genetic technique by which an unknown plasmid can be classified by the use of plasmid of known incompatibility group . In phenocopy state, cells harboring plasmids of known incompatibility group behave as normal recipients and receive plasmids belonging to the same incompatibility group at a high frequency . This work provides additional evidence that plasmids in 'phenocopy' hosts do not replicate and therefore fail to demonstrate their incompatibility barrier to the incoming plasmids . A cryptic plasmid, now called pWS7, has been identified by this method in a pili, fla female strain of E . coli K12 . Genetic analysis shows the plasmid pWS7 is in fact, a sex-factor which is curable with acridine orange . It belongs to the Inc F1 group . Physical analysis confirms its size to be 124 Kb . The plasmid has been labelled genetically with a transposon Tn903 in a recA host and further characterized by heteroduplex analysis . A DNA sequence homology between pWS7 and F'lac plasmid extends only in F-regions, 2.8F-94.5F . The pili, fla host strain of pWS7 shows a high frequency of transformation for recombinant DNA and rapid propagation for a male-specific RNA phage, R17.

Infection, 1982, 10(2), 112 - 5
Operon fusion of the phase variation switch . A virulence factor in Escherichia coli; Eisenstein BI; A derivative strain of a K-12 isolate of Escherichia coli was selected utilizing the operon fusion technique of Casadaban . This derivative strain (VL 361) was characterized by its ability to synthesize the enzyme beta-galactosidase - the gene product of the inserted lacZ gene - as if it were type 1 fimbriae . Enzyme production was found to behave according to the properties of phase variation, thus mimicking the phase variation seen with type 1 fimbriae . The oscillation between the on-and-off synthesis of enzyme was found to be random, and was not influenced by such environmental factors as temperature, glucose or cyclic AMP . Thus the selection in various media of either fimbriate or non-fimbriate states is probably not genetic in nature, but rather the outgrowth of one type of phase over another.

Mol Biochem Parasitol, 1981 Dec 31, 4(5-6), 273 - 82
Genome organization and ploidy number in Trypanosoma cruzi; Castro C et al.; Trypanosoma cruzi total DNA was analyzed by DNA:DNA reassociation kinetics . The nonlinear least-squares computer solution could reasonably be fitted to three second-order kinetic components . The highly repetitive, middle repetitive, and single copy components comprised 9, 35 and 49% of the genome, respectively . The single copy sequences showed a kinetic complexity of approximately 4 times that of Escherichia coli and of some 11,000 average-sized structural genes . The repetitive sequences (about 6900) presented the long-period pattern of interspersion with a modal length of 7800 bases . The kinetic complexity of total DNA was compatible with a value of at least a diploid genome per cell.

Biochim Biophys Acta, 1981 Dec 28, 656(2), 240 - 5
Purification and properties of fMet-tRNAf deacylase from Escherichia coli; Igarashi K et al.; A formylmethionyl-tRNAf deacylase has been purified about 330-fold from a crude initiation factor preparation (1 M NH4Cl ribosomal wash) from Escherichia coli Q13 . The enzyme was nearly homogeneous and had an apparent molecular weight of 24 000 . Rat liver methionyl-tRNAf and E . coli methionyl-tRNAm were not hydrolyzed significantly by the enzyme under standard conditions . Q beta RNA- and AUG(A)n-directed polypeptide synthesis was inhibited by the enzyme . The inhibition was at the level of initiation of polypeptide synthesis . The enzymatic activity was inhibited by various factors necessary for polypeptide synthesis . The activity was inhibited more by NH4Cl and spermidine than by Mg2+, GTP and ATP . The complex of formylmethionyl-tRNAf, initiation factor 2 and GTP was resistant to enzymatic hydrolysis, and the resistance was enhanced by the addition of AUG and ribosomes to the above reaction mixture.

Biochim Biophys Acta, 1981 Dec 28, 656(2), 140 - 6
The effect of ATP on the incorporation of deoxyribonucleoside triphosphates by Escherichia coli DNA polymerase I; Spasokukotskaja T et al.; The effect of ATP on Escherichia coli DNA polymerase I has been investigated as a function of the concentration of substrates and divalent metal ions in the presence of activated DNA as template . At saturating Mg2+ concentration 1.5 mM ATP stimulated 2.5-times the incorporation of {3H}dGTP when only one substrate (dGTP) was present, and had no significant effect in the presence of all four dNTPs, whereas under similar conditions, a saturating concentration of dATP increased the reaction rate only 1.5-times . At optimal Mn2+ concentrations ATP also showed a similarly marked effect only in the case when one substrate (dGTP) was present in the reaction . The optimal concentration of Mn2+ was shifted by ATP to higher concentrations both in the presence of one and of all four substrates . ATP did not influence the apparent Km for dGTP, while V was increased by a factor of about 2.5 . The possible presence of dNTP in ATP, as inpurity, was ruled out by isotope dilution analysis . Thus, ATP stimulated the polymerization reaction only under limited conditions, i.e., when one substrate was present in the reaction.

Biochim Biophys Acta, 1981 Dec 28, 656(2), 134 - 9
Differential stimulation by polyamines of phage RNA-directed synthesis of proteins; Watanabe Y et al.; The effect of polyamines on Q beta and MS2 phage RNA-directed synthesis of three kinds of protein in an Escherichia coli cell-free system has been studied . With both phage RNAs, the degree of stimulation of protein synthesis by spermidine was in the order RNA replicase greater than A protein, while the synthesis of coat protein was not stimulated significantly by spermidine . The synthesis of RNA replicase was stimulated by 1 mM spermidine approx . 8-fold . From the results of Q beta RNA direct alanyl-tRNA and seryl-tRNA binding to ribosomes and initiation dipeptide synthesis, it is suggested that the preferential stimulation of the synthesis of RNA replicase by spermidine is due at least partially to the stimulation of the initiation of RNA replicase synthesis.

Biochim Biophys Acta, 1981 Dec 28, 656(2), 189 - 94
In vitro conversion of S13 viral DNA in phage particles to the double-stranded DNA; Watabe K et al.; The conversion of single-stranded DNA in S13 intact phage particles to the double-stranded replicative form DNA was observed in cell extracts prepared from Escherichia coli H560 (S13s, polA, endA) cells lysed with lysozyme and the non-ionic detergent, Brij 58 . The DNA product, which associated with a rapidly sedimenting component, was identified as RFII-DNA with a gap by sedimentation analysis . The conversion was inhibited by N-ethylmaleimide, but not by rifampicin, nicotinamide mononucleotide or polymyxin B . The dnaB gene product was involved in the replicative system . Similar extracts prepared from a S13-resistant E . coli strain K12W6 also catalyzed this synthesis.

J Biol Chem, 1981 Dec 25, 256(24), 13218 - 22
The radius of gyration of L-arabinose-binding protein decreases upon binding of ligand; Newcomer ME et al.; The technique of small angle x-ray scattering has been employed to study the effect of sugars on the radius of gyration of the L-arabinose-binding protein, a component of the high affinity L-arabinose transport system in Escherichia coli . We find that the binding of L-arabinose to the "sugar-free" protein in solution causes a 0.94 +/- 0.33 A decrease in the radius of gyration while D-glucose, a nonbinder, produces no such effect . The radius of gyration calculated from the complete atomic co-ordinates of the crystal structure of L-arabinose-binding protein (solved with bound L-arabinose) corresponds to the experimentally determined value for the radius of gyration in the presence of L-arabinose . This reduction in radius of gyration can be best accounted for in terms of a substrate-induced cleft closure in which one lobe rotates relative to the other lobe . A compute modeling study indicates that a rotation of 18 degrees about a hinge deep in the base of the sugar-binding cleft between the two domains would produce the observed decrease in the radius of gyration . The findings (Newcomer, M . E., Gilliland, G . L., and Quiocho, F . A . (1981) J . Biol . Chem . 256, 13213-13222) that the L-arabinose molecule embedded in the cleft between two domains is completely inaccessible to the solvent is consistent with a closing of the cleft between the two lobes.

J Biol Chem, 1981 Dec 25, 256(24), 13213 - 7
L-Arabinose-binding protein-sugar complex at 2.4 A resolution . Stereochemistry and evidence for a structural change; Newcomer ME et al.; The L-arabinose molecule (in the C1 pyranose chair conformation) has been fitted to the electron density corresponding to the bound sugar in the 2.4 A resolution Fourier map of the L-arabinose-binding protein . The sugar molecule is buried in the cleft between the two lobes of the bilobate protein . All sugar hydroxyls are hydrogen-bonded to side chain residues: beta-OH(1) to Lys-10 and Asp-90, OH(2) to Lys-10, OH(3) to Asn-205 and Glu-14 (possibly via a water molecule), and OH(4) to Asn-232 . Lys-10, Glu-14, and Asp-90 are associated with one domain while Asn-205 and Asn-232 are lodged in the other . Protein structural change accompanying binding is indicated by the inaccessibility of the bound L-arabinose to the aqueous environment.

J Biol Chem, 1981 Dec 25, 256(24), 12836 - 9
A sensitive immunoblotting method for measuring protein synthesis initiation factor levels in lysates of Escherichia coli; Howe JG et al.; Protein synthesis initiation factor levels are measured in crude cell lysates of Escherichia coli MRE600 by use of a sensitive immunoblotting method . The method involves electrophoretic transfer of protein from sodium dodecyl sulfate-polyacrylamide gels onto nitrocellulose paper and subsequent incubation with a specific antiserum and radioactive iodinated second antibody . The measurement of iodinated antibody attached to known amounts of initiation factor is determined by densitometric scanning of autoradiographs or counting radioactivity in excised protein bands . Linear standard curves over the range 1 to 300 ng of factor are obtained by these methods . Unknown amounts of initiation factor in crude cell lysates are measured accurately; values agree with previous radioimmune assay data . The immunoblotting method serves as an alternative to the radioimmune assay in measuring small quantities of protein in complex mixtures . Immunoblotting enjoys three major advantages: it is simple and rapid to execute; it is sensitive; and it is capable of distinguishing multiple forms of the antigen which separate in the gel system employed.

J Biol Chem, 1981 Dec 25, 256(24), 13200 - 6
DNA determinants important in sequence recognition by Eco RI endonuclease; Lu AL et al.; Alkylation interference and protection methods (Siebenlist, U., and Gilbert, W., (1980) Proc . Natl . Acad . Sci . U . S . A . 77, 122-126) have been utilized to deduce potential DNA contacts involved in specific complex formation between Eco RI endonuclease and its recognition sequence . The endonuclease protected the N7 position (major groove) of the dG and the N3 position (minor groove) of both dA residues within the Eco RI sequence against alkylation by dimethylsulfate, d(GpApApTpTpC), suggesting the presence of poly-peptide in both grooves in the vicinity of affected nitrogens . Results of methylation interference analysis suggest that the N7 of the Eco RI site dG and the N3 of the central dA, d(GpApApTpTpC), are utilized as contacts by the enzyme . The failure to observe interference upon methylation of the 5'-penultimate dA within the sequence implies that the endonuclease does not bond to the N3 of this residue, despite the fact that it is protected against alkylation by the protein . Ethylation interference patterns suggest four major phosphate contacts between endonuclease and each DNA strand . Two of these phosphates are 5'-external to the Eco RI sequence, d(pNpGpApApTpTpC), suggesting involvement of outside phosphates in electrostatic interactions . Moreover, alkylation protection and interference effects on the two DNA strands display perfect 2-fold symmetry . Thus, the endonuclease interacts with a minimum of 10 nucleotide pairs to yield a DNA-protein complex characterized by elements of symmetry . In contrast, specific alkylation effects were not observed in comparable experiments with the endonuclease and a DNA which had been previously methylated by the Eco RI modification enzyme.

Biochemistry, 1981 Dec 22, 20(26), 7539 - 46
Characteristics of beta, beta-difluoroalanine and beta, beta, beta -trifluoroalanine as suicide substrates for Escherichia coli B alanine racemase; Wang EA et al.; The alanine racemase from Escherichia coli B has been shown to process DL isomers of beta -fluoroalanine as suicide substrates with an identical partitioning ratio for each enantiomer of 820 catalytic eliminations of HF per enzymatic inactivation event {Wang, E., & Walsh, C . T . (1978) Biochemistry 17, 1313}, suggesting the aminoacrylate--PLP complex as a common, symmetrical partitioning species . In an attempt to vary the partition ratio, an index of killing efficiency, systematically the beta, beta-difluoroalanine and beta, beta, beta-trifluoroalanine isomers have now been evaluated for substrate processing, suicidal inactivation kinetics and partitioning ratio, and stability of inactive, derivatized enzyme forms . Both difluoroalanine isomers show high Km values (116 mM for D, 102 mM for L) in catalytic HF loss to form fluoropyruvate . The Vmax for the D isomer is about 14-fold higher than that for the L isomer . Limiting inactivation rate constants, calculated from kcat and observed partition ratios of 5000 and 2600, respectively, are 2.2 min-1 for D-difluoroalanine and 0.33 min-1 for L-difluoroalanine . For comparison, DL-trifluoroalanine turns over less than 10 times per enzyme molecule inactivated and so is a very efficient suicide substrate . The estimated inactivation rate constant is less than or equal to 1.0 min-1 . These data are analyzed in terms of partitioning behavior of the monofluoro- and difluoroaminoacrylate--PLP complexes as partitioning intermediates for turnover or for racemase inactivation . While mono- and trifluoroalanines yield stable inactive species, the difluoroalanine isomers produce labile enzyme derivatives, and regain of catalytic activity is analyzed in terms of the anticipated oxidation state at the beta carbon of the substrate fragment adducted to the enzyme.

Biochemistry, 1981 Dec 22, 20(26), 7494 - 501
Steady-state and pre-steady-state kinetics of coenzyme A linked aldehyde dehydrogenase from Escherichia coli; Shone CC et al.; Coenzyme A linked aldehyde dehydrogenase from Escherichia coli strain B has been purified to a specific activity of 14 units/mg of protein, and initial rate and substrate analogue inhibition experiments have been performed . On the basis of these steady-state measurements, a bi-uni-uni-uni ping-pong mechanism is proposed in which NAD+ binds to the free enzyme followed by acetaldehyde . The product NADH is then released before coenzyme A (CoA) can bind, and acetyl-CoA is the final product released . A pre-steady-state time-dependent activation of the enzyme was observed when assays were initiated with NAD+ . This lag phase of the reaction was studied as a function of the NAD+ concentration and found to be first order . Furthermore, the presence of NAD+ was demonstrated to be necessary to maintain the enzyme in the active conformation . Evidence that the enzyme contains two distinct NAD+ binding sites, an activator site and a catalytic site, has been obtained from pre-steady-state experiments with the NAD+ analogues AMP and 3-pyridine-carboxaldehyde adenine dinucleotide . AMP, a potent competitive inhibitor with respect to NAD+ under steady-state conditions, did not affect the rate of enzyme activation during pre-steady-state measurements . The analogue 3-pyridine-carboxaldehyde adenine dinucleotide, a potent activator of the aldehyde dehydrogenase, was a poor substrate compared with NAD+ . At concentrations of this analogue that fully activated the enzyme, no alternate substrate inhibition was observed with respect to NAD+ . A model incorporating two binding sites for NAD+ has been put forward to explain these observations.

Biochemistry, 1981 Dec 22, 20(26), 7519 - 28
Modification of the allosteric activator site of Escherichia coli ADP-glucose synthetase by trinitrobenzenesulfonate; Carlson CA et al.; Limited modification of Escherichia coli B ADP-glucose synthetase (EC 2.7.7.27) by trinitrobenzenesulfonate (TNBS) appeared to affect primarily the allosteric properties of the enzyme . There was little loss of the catalytic activity assayed in the absence of activator . However, the abilities of fructose 1,6-bisphosphate or hexanediol 1,6-bisphosphate to activate the enzyme, or of 5'-adenylate to inhibit the enzyme, were rapidly lost upon trinitrophenylation . Modification progressively decreased the affinity for activator, decreased the Vmax at saturating concentrations of activator, and decreased the cooperativity among activator binding sites . These effects could be completely prevented by the presence of allosteric effectors during reaction with TNBS, although a low amount of trinitrophenylation still occurred . Substrates partially protected the enzyme from reaction with TNBS . The lysyl epsilon-amino side chain was modified by trinitrophenylation, but the target was not primarily the same residue which could form a Schiff base with pyridoxal phosphate, another activator of the enzyme . A large peptide containing most of the trinitrophenyl residue was isolated after cleavage of the enzyme and was identified as part of the N-terminal amino acid sequence . The migration of the enzyme on polyacrylamide gel electrophoresis or on agarose column chromatography was unchanged by modification . However, the ability of fructose-1, 6-P2 to induce the oligomerization of a mutant form of the enzyme was completely prevented by trinitrophenylation . This effect could be protected against by the presence of activator or inhibitor during reaction with TNBS.

Nucleic Acids Res, 1981 Dec 21, 9(24), 6985 - 94
(E)-5-(2-bromovinyl)-2'-deoxyuridine-5'-triphosphate as a DNA polymerase substrate; Sagi J et al.; Time course of incorporation and the effect of 5'-triphosphate of the selective antiherpetic agent (E)-5-(2-bromovinyl)-2'-deoxyuridine (bv5dUTP) on the incorporation of dTTP and dATP into template-primers of different structure were studied in E . coli DNA polymerase I Klenow fragment enzyme-catalyzed reactions . bv5dUTP could substitute for dTTP depending on the structure of template-primer . E.g . into calf thymus DNA incorporation of bv5dUMP was around 80% of that of dTMP at 30 minutes of incubation . The analog has also inhibited dTMP incorporation, net DNA synthesis, however, was hardly affected . The substrate properties of the analog were studied with {2-14C}-labelled bv5dUTP.

Nucleic Acids Res, 1981 Dec 21, 9(24), 6935 - 52
The primary and secondary structure of yeast 26S rRNA; Veldman GM et al.; We present the sequence of the 26S rRNA of the yeast Saccharomyces carlsbergensis as inferred from the gene sequence . The molecule is 3393 nucleotides long and consists of 48% G+C; 30 of the 43 methyl groups can be located in the sequence . Starting from the recently proposed structure of E . coli 23S rRNA (see ref . 25) we constructed a secondary structure model for yeast 26S rRNA . This structure is composed of 7 domains closed by long-range base pairings as n the bacterial counterpart . Most domains show considerable conservation of the overall structure; unpaired regions show extended sequence homology and the base-paired regions contain many compensating base pair changes . The extra length of the yeast molecule is due to a number of insertions in most of the domains, particularly in domain II . Domain VI, which is extremely conserved, is probably part of the ribosomal A site . alpha-Sarcin, which apparently inhibits the EF-1 dependent binding of aminoacyl-tRNA, causes a cleavage between position 3025 and 3026 in a conserved loop structure, just outside domain VI . Nearly all of the located methyl groups, like in E . coli, are present in domain II, V and VI and clustered to a certain extent mainly in regions with a strongly conserved primary structure . The only three methyl groups of 26S rRNA which are introduced relatively late during the processing are found in single stranded loops in domain VI very close to positions which have been shown in E . coli 23S rRNA to be at the interface of the ribosome.

Nucleic Acids Res, 1981 Dec 21, 9(24), 6647 - 68
The complete nucleotide sequence of the tryptophan operon of Escherichia coli; Yanofsky C et al.; The tryptophan (trp) operon of Escherichia coli has become the basic reference structure for studies on tryptophan metabolism . Within the past five years the application of recombinant DNA and sequencing methodologies has permitted the characterization of the structural and functional elements in this gene cluster at the molecular level . In this summary report we present the complete nucleotide sequence for the five structural genes of the trp operon of E . coli together with the internal and flanking regions of regulatory information.

Biochim Biophys Acta, 1981 Dec 21, 649(3), 661 - 79
Lactose-H+(-OH) transport system of Escherichia coli . Multistate gated pore model based on half-sites stoichiometry for high-affinity substrate binding in a symmetrical dimer; Lombardi FJ; A model is proposed for the D-galactoside-H+(-OH) transporter of Escherichia coli that accounts for essentially all the experimental observations established for this system to date . In this model, the functional unit is postulated to be a dimer (consisting of two copies of lac Y-specified polypeptide) which spans the membrane with a 2-fold symmetry axis in the membrane plane (Lancaster, J.R . (1978) J . Theor . Biol . 75, 35-50) . The functional dimer is assumed to possess a single pore flanked by an inner gate (gi) and an outer gate (go) and encompassing two oppositely oriented galactoside binding sites, designated m and mu . When go is open and gi is closed under non-energized conditions, binding site m adopts a configuration defined as State A (i.e., moA) exhibiting high affinity toward Class Ga galactosides (thiodigalactoside, melibiose, alpha-p-nitrophenygalactoside) but low affinity for Class Gb galactosides (lactose, beta-o-nitrophenylgalactoside, beta-isopropylthiogalactoside), whereas binding site mu adopts State B (i.e., muoB) displaying relatively high affinity toward Class Gb galactosides but comparatively low affinity for Class Ga galactosides; further, each moA : muoB dimer contains one thiol group whose reaction with N-ethylmaleimide inactivates the transporter unless blocked by galactoside binding at site moA, while the second homologous thiol of the dimer is unreactive toward thiol reagents . Translocation of the moA : muoB dimer involves closing of go followed by opening of gi, and causes the two thiols (as well as sites m and mu) to interchange roles in a symmetrical fashion: moA : muoB in equilibrium miB : muiA . In the presence of a substantial (negative) transmembrane delta potential of muH+, the m : mu dimer is postulated to undergo an electrogenic protein conformation change to a second form, *(m : mu), in which both sites m and mu possess low affinity toward internal Class Gb substrates; galactoside transport in both m : mu and *(m : mu) is assumed to be coupled to H+-symport (-OH-antiport) with a stoichiometry of approximately 1 : 1 . Finally, five characteristic predictions of the half-sites model are outlined for further tests of its validity.

Nucleic Acids Res, 1981 Dec 21, 9(24), 6827 - 40
Nucleotide sequence at the end of the gene for the RNA polymerase beta' subunit (rpoC); Squires C et al.; We have determined the DNA sequence surrounding the transcription terminator following rpoC, the gene that codes for the beta' subunit of RNA polymerase in E . coli K12 . The 2044 bp sequence obtained contains the distal 335 codons of rpoC followed by a 212 bp non-coding region and a second open reading frame (ORFa) of 179 codons . The final 181 nucleotides of the sequence form the 5' end of a third open reading frame (ORFb) . The in vivo 3' end of the rpoC mRNA was located by analysis of RNA/DNA hybrids cleaved with nuclease S1 (S1 mapping) . These results indicated that the major transcription termination of the rplJL-rpoBC transcription unit occurs a short distance past the translation stop codon for rpoC . Four regions of symmetry, suggesting secondary structure in the mRNA, were found in the DNA sequence near the rpoC translation termination codon . The last of these hairpin structures is similar to other rho-independent transcription terminators and its 3' end coincides with the end of the rpoC mRNA as predicted by S1-mapping . Inspection of the open reading frames indicates that rpoC uses a high percentage of codons that are recognized by the major tRNA species of E . coli while ORFa and ORFb contain many codons recognized by minor tRNA species . ORFa specifies a very basic peptide.

Biochem J, 1981 Dec 15, 200(3), 611 - 27
Energization of the transport systems for arabinose and comparison with galactose transport in Escherichia coli; Daruwalla KR et al.; 1 . Strains of Escherichia coli were obtained containing either the AraE or the AraF transport system for arabinose . AraE+,AraF- strains effected energized accumulation and displayed an arabinose-evoked alkaline pH change indicative of arabinose-H+ symport . In contrast, AraE-,AraF+ strains accumulated arabinose but did not display H+ symport . 2 . The ability of different sugars and their derivatives to elicit sugar-H+ symport in AraE+ strains was examined . Only L-arabinose and D-fucose were good substrates, and arabinose was the only inducer . 3 . Membrane vesicles prepared from an AraE+,AraF+ strain accumulated the sugar, energized most efficiently by the respiratory substrates ascorbate + phenazine methosulphate . Addition of arabinose or fucose to an anaerobic suspension of membrane vesicles caused an alkaline pH change indicative or sugar-H+ symport on the membrane-bound transport system . 4 . Kinetic studies and the effects of arsenate and uncoupling agents in intact cells and membrane vesicles gave further evidence that AraE is a low-affinity membrane-bound sugar-H+ symport system and that AraF is a binding-protein-dependent high-affinity system that does not require a transmembrane protonmotive force for energization . 5 . The interpretation of these results is that arabinose transport into E . coli is energized by an electrochemical gradient of protons (AraE system) or by phosphate bond energy (AraF system) . 6 . In batch cultures the rates of growth and carbon cell yields on arabinose were lower in AraE-,AraF+ strains than in AraE+,AraF- or AraE+,AraF+ strains . The AraF system was more susceptible to catabolite repression than was the AraE system . 7 . The properties of the two transport systems for arabinose are compared with those of the genetically and biochemically distinct transport systems for galactose, GalP and MglP . It appears that AraE is analogous to GalP, and AraF to MglP.

Biochem J, 1981 Dec 15, 200(3), 583 - 9
The effects of partial and selective reduction in the components of the proton-motive force on lactose uptake in Escherichia coli; Ahmed S et al.; The relationship between the steady state lactose accumulation (delta plac) and the magnitude of the membrane potential (delta psi) and pH gradient (delta pH) has been studied at pHo5.5 and pHo7.5 . An attempt has been made to differentiate between two possible means by which lactose accumulation may be reduced below the proton-motive force (delta p) . Firstly, that delta psi and delta pH are not equivalent in driving lactose transport and secondly, that 'slip' reactions (beta-galactoside exit via the carrier without a proton) may reduce accumulation . The data support the latter; however, our conclusions are tempered by the observation that the apparent stoichiometry (delta plac/delta p) increases to a value of at least 2 at values of delta p below 130 mV.

Biochem J, 1981 Dec 15, 200(3), 573 - 81
Quantitative measurements of the proton-motive force and its relation to steady state lactose accumulation in Escherichia coli; Ahmed S et al.; The magnitude of delta psi (membrane potential), delta pH (pH gradient), lactose accumulation and cytoplasmic volume have been determined over a range of experimental conditions . A study of two probes of delta pH, benzoate and dimethyloxazolidene-2,4-dione (DMO), and four probes of delta psi, Rb+, K+, tetraphenylphosphonium (TPP+) and 3,3'-dipropylthiodicarbocyanine iodide, has been carried out . Benzoate and DMO are shown to be equivalent at pH values above the pK of DMO, but the latter may be less accurate below this pH . The cations TPP+ and Rb+ were found, by a number of criteria, to be equivalent, and TPP+ may be used in cells not pretreated with EDTA . These studies are an essential prerequisite to the use of TPP+ as a quantitative probe in untreated cells.

Nucleic Acids Res, 1981 Dec 11, 9(23), 6553 - 69
1H NMR studies of lac-operator DNA fragments; Zuiderweg ER et al.; The hydrogen-bonded imino protons of a 14 base pair double-stranded DNA fragment comprising one half of the lac operator of E . coli were investigated by 360 MHz H NMR . From combined melting studies of this synthetic 14 b.p . fragment and its two constituent 7 b.p . fragments a nearly complete assignment for the low-field proton resonances was obtained . The experimental spectra are compared with calculated spectra and with the spectrum of a 51 b.p . DNA restriction fragment from E . coli containing the complete lac operator . Structural information on these oligonucleotides is presented . This study is a prerequisite for future 1H NMR investigations of the interaction of the lac operator with the lac repressor.

Nucleic Acids Res, 1981 Dec 11, 9(23), 6421 - 8
Primary structure of an unusual glycine tRNA UGA suppressor; Prather NE et al.; We have determined the nucleotide sequences of two UGA-suppressing glycine transfer RNAs . The suppressor tRNAs were previously shown to translate both UGA and UGG and to have arisen as a consequence of mutation in glyT, the gene for the GGA/G-reading glycine tRNA of Escherichia coli . In each mutant tRNA, the primary sequence change was the substitution of adenine for cytosine in the 3' position of the anticodon . In addition, a portion of mutant glyT tRNA molecules contained N6-(delta 2-isopentenyl)-2-thiomethyl adenine adjacent to the 3' end of the anticodon (nucleotide 37) . The presence or absence of this hypermodification may be a determinant in some of the biological properties of the mutant tRNA.

JAMA, 1981 Dec 11, 246(23), 2737 - 9
Gene therapy; Anderson WF; KIE: Guidance is provided for the practicing physician confronted with patients inquiring about the applicability and potential benefits of gene therapy . The state of the art and the limitations of present techniques are reviewed, and general criteria for potential use of the procedures in humans are supplied . While gene therapy holds future promise for treatment of single gene defects, the technology has not evolved to the point where it should now be used in patient care .

Nucleic Acids Res, 1981 Dec 11, 9(23), 6487 - 503
Characterization and mapping of RNase III cleavage sites in VSV genome RNA; Wertz GW et al.; Ribonuclease III cleaves the genome RNA of vesicular stomatitis virus (VSV) to yield an array of fragments which range in size from 3.5 to 0.1 x 10(6) daltons under partial digestion conditions . The locations of the RNase III cleavage sites which give rise to these fragments have been ordered relative to the 3' end of the virion RNA by digestion of 3' end-labeled RNA . Based on a map of the cleavage sites we predicted that fragments having the same size could be generated which contain information from each gene . Annealing of individual VSV mRNA probes to Northern blots of the separated RNase III-generated fragments confirmed that fragments having the same size are, in fact, generated which contain information from each coding region of the VSV genome . Analysis of maps of partial digestion products indicates that fragments having the same size arise repeatedly along the 3' half of the genome . The cleavage of VSV RNA by RNase III can be detected only if the nuclease treated molecules are denatured . This suggest that the structure features in VSV RNA which signal cleavage involve areas of higher order RNA structure.

Nucleic Acids Res, 1981 Dec 11, 9(23), 6391 - 406
Structure and sequence of the mitochondrial 20S rRNA and tRNA tyr gene of Paramecium primaurelia; Seilhamer JJ et al.; The sequence and structure of the large (20s) mitochondrial (mt) rRNA gene and flanking regions from Paramecium primaurelia have been determined . The gene contains two regions of strong homology with other large mt rRNAs: one 44-base region near the 5' end and a 321-base region near the 3' end . Another region of strong homology to both ends of E . coli 23s RNA exists at loci consistent with these regions . The Paramecium gene appears to be 2204 bases in length and contains slightly more homology to E . coli rRNA than its mammalian or fungal counterparts . The gene, located about 1200 bp from the replicative terminal end of the linear mt DNA, is transcribed in the same polarity as replication . Previous R-looping studies detected no large introns within the gene . Here we describe sequences resembling degenerate rRNAs, one of which could represent a small intron . A tRNA tyr gene was found on the same DNA strand, 127 bp downstream from the large rRNA presumptive 3' end . The tRNA is flanked on both sides by short DNA regions of approximately 90% A + T content.

J Biol Chem, 1981 Dec 10, 256(23), 11988 - 91
The reconstitution of binding protein-dependent active transport of glutamine in isolated membrane vesicles from Escherichia coli; Hunt AG et al.; The reconstitution of binding protein-dependent glutamine transport in isolated membrane vesicles from Escherichia coli is described . The reconstituted glutamine transport is shown to be energy-dependent and does not involve the metabolism of glutamine or the trapping of liganded binding protein with the vesicles . The preparation of vesicles capable of transporting glutamine in a binding protein-dependent manner required the incorporation of NAD into vesicles and the use of a binding protein point mutant as the source of the vesicles.

Nature, 1981 Dec 10, 294(5841), 536 - 40
Clustered arrangement of immunoglobulin lambda constant region genes in man; Hieter PA et al.; The immunoglobulin lambda light chain locus of man contains six lambda-like genes arranged tandemly on a 50-kilobase segment of chromosomal DNA . THe sequences of three of these genes correspond to three known non-allelic lambda chain isotypes: Mcg, Ke-Oz- and Ke-Oz+ . They surround a highly polymorphic and evidently unstable region that is repeatedly deleted when cloned in Escherichia coli . Three additional, but as yet unlinked, lambda-like sequences have also been cloned, suggesting that the lambda genes form an unexpectedly large family within the human genome.

J Biol Chem, 1981 Dec 10, 256(23), 12545 - 52
Cyclic AMP regulation of lactate dehydrogenase . Quantitation of lactate dehydrogenase M-subunit messenger RNA in isoproterenol-and N6,O2'-dibutyryl cyclic AMP-stimulated rat C6 glioma cells by hybridization analysis using a cloned cDNA probe; Miles MF et al.; We have cloned DNA complementary to mRNA coding for rat C6 glioma cell lactate dehydrogenase M-subunit . Double-stranded DNA complementary to a portion of lactate dehydrogenase mRNA was inserted into the Pst I site of plasmid pBR322 by the dC.dG tailing technique and amplified in Escherichia coli HB101 . A recombinant plasmid containing lactate dehydrogenase cDNA was identified by colony hybridization to a cDNA prepared from partially purified lactate dehydrogenase mRNA and by hybridization-selected translation . The recombinant plasmid (pRLD42) contains a 680 nucleotide insert of lactate dehydrogenase mRNA . Hybridization of nick-translation pRLD42 to glioma cell poly(A)+RNA separated on agarose gel and transferred to nitrocellulose exhibited Mr = 5.9 X 10(5) for lactate dehydrogenase mRNA . Furthermore, Northern blot analysis of RNA from unstimulated and isoproterenol-stimulated glioma cells indicated a 2-fold increase of lactate dehydrogenase mRNA molecules in stimulated cells . The 2-fold increase of lactate dehydrogenase mRNA was confirmed by RNA-excess kinetic hybridization using pRLD42 DNA and poly(A)+RNA from unstimulated, isoproterenol-, and dibutyryl cAMP-stimulated glioma cells . These data demonstrate that isoproterenol and dibutyryl cAMP cause an increase of the number of lactate dehydrogenase M-subunit mRNA molecules in glioma cells which, in part, determines the extent of synthesis of the lactate dehydrogenase M-subunit.

J Biol Chem, 1981 Dec 10, 256(23), 12301 - 5
The binding site for ribosomal protein L11 within 23 S ribosomal RNA of Escherichia coli; Schmidt FJ et al.; Ribosomal protein L11 of Escherichia coli was bound to 23 S rRNA and the resultant complex was digested with ribonuclease T1 . A single RNA fragment, protected by protein L11, was isolated from such digests and was shown to rebind specifically to protein L11 . The nucleotide sequence of this RNA fragment was examined by two-dimensional fingerprinting of ribonuclease digests . It proved to be 61 residues long and the constituent oligonucleotides could be fitted perfectly between residues 1052 and 1112 of the nucleotide sequence of E . coli 23 S rRNA.

J Biol Chem, 1981 Dec 10, 256(23), 11970 - 3
Stabilization by ATP and ADP of Escherichia coli dnaB protein activity; Gunther E et al.; The effect of adenine ribonucleotides on the stability of Escherichia coli dnaB protein in cellular crude extracts was studied . Stabilization of dnaB protein by ATP or ADP, but not by AMP, was manifested in that (i) the activity and yield of wild type dnaB protein is enhanced in the presence of ATP, (ii) the dnaB protein of E . coli dnaB mutants, such as groPB and dnaB252/ColE1::dnaC+, which is inactive in a dnaB complementation assay, can be isolated in active form in the presence of ATP or aDP, (iii) ATP or ADP protect the dnaB protein of an E . coli dnaBts mutant from inactivation at 37 degrees C, and (iv) inactive groPB and dnaBts protein can be reactivated partially by ATP . Thus, the stabilizing effect of ATP and ADP can be exploited for the isolated of otherwise inactive or labile mutant dnaB proteins.

Clin Chim Acta, 1981 Dec 9, 117(2), 199 - 207
Highly sensitive sandwich enzyme immunoassay of human IgE with beta-D-galactosidase from Escherichia coli; Imagawa M et al.; A highly sensitive sandwich enzyme immunoassay of human IgE was developed . Polystyrene balls were coated with goat anti-human IgE immunoglobulin (IgG) by physical adsorption . Goat anti-human IgE Fab' was purified by affinity chromatography and conjugated with beta-D-galactosidase from Escherichia coli . Using thus prepared anti-IgE-coated polystyrene balls and anti-IgE-beta-D-galactosidase conjugate, 0.2 mU (2 amol)--1 U of IgE per assay could be determined . When 0.1 microliter of serum per assay was used, the range of IgE levels in serum that could be determined was 2--10000 U/ml, and even 0.01 U/ml was measurable by using 20 microliters of serum . The regression equation and coefficient for correlation to radioimmunoassay were gamma (RIA) = 0.94 chi (EIA) + 18.2 and 0.96 (n = 81), respectively . The coefficients of within- and between-assay variations ranged from 5.4 to 8.5% . The mean levels of serum IgE determined by the present assay were 103 U/ml in 70 normal children and 1064 U/ml in 38 children with bronchial asthma.

Biochemistry, 1981 Dec 8, 20(25), 7295 - 301
Optically detected magnetic resonance of the phosphorescent bases of Escherichia coli valine-specific transfer ribonucleic acid; Taherian MR et al.; Phosphorescence spectroscopy and optical detection of triplet state magnetic resonance (ODMR) spectroscopy have been used to characterize bases that contribute to the phosphorescence emission of Escherichia coli valine-specific transfer ribonucleic acid . When it is excited with 335-nm light, a short-lived phosphorescence with an origin near 435 nm is observed and is assigned to 4-thiouridine (s4U) at position 8 of the tRNA sequence . With excitation at 290-300 nm, a structured, long-lived phosphorescence is observed with an origin near 380 nm, in addition to the s4U phosphorescence . Comparison was made of the phosphorescence and ODMR spectra between Mg2+-containing and Mg2+-free tRNA samples . The s4U phosphorescence of the Mg2+-containing sample is more structured, and the peak is blue shifted relative to the Mg2+-free sample . Both samples give a single low-frequency (ca . 2.9 GHz) ODMR signal, but the high-frequency signal region (ca . 19-20 GHz) is structured . The Mg2+-containing sample has a partially resolved group of lines centered at 19.3 GHz, whereas the Mg2+-free sample has two broad bands centered at 19.2 and 20.0 gHz . The differences are attributed to effects of Mg2+ on the tRNA conformation . The ODMR signals observed by monitoring the long-lived phosphorescence are assigned to a pyrimidine nucleoside, possibly 5-(carboxy-methoxy)uridine in the anticodon.

Biochemistry, 1981 Dec 8, 20(25), 7059 - 64
Discrimination between D- and L-tyrosyl transfer ribonucleic acids in peptide chain elongation; Yamane T et al.; D-Tyr-tRNA can take part in peptide bond formation with N-AcPhe-tRNA on ribosomes programmed with the hexanucleotide UUUUAC . None of the steps leading to peptide bond formation exhibit high stereoselectivity . Ternary complex formation with EF-Tu.GTP favors L-Tyr-tRNA by a factor greater than 25 . The complex formed with D-Tyr-tRNA was not protected from hydrolysis, which suggests that the D-amino acid is improperly bound to the protein . The rate of EF-Tu-promoted dipeptide formation was 30-fold faster with L-Tyr-tRNA . The ratio of moles of GTP hydrolyzed to dipeptide formed was 1.4 for L-Tyr-tRNA and 4 for D-Tyr-tRNA . The excess of GTP hydrolyzed to peptide bonds formed is evidence for kinetic proofreading in AA-tRNA selection . The combined effects of the partial discrimination at each stage, from the aminoacylation to the peptide formation, favor L-tyrosine by a factor greater than 10(4) and would virtually exclude D-tyrosine from being incorporated under conditions where L-tyrosine was also present.

Biochim Biophys Acta, 1981 Dec 7, 649(2), 419 - 26
Counterflow efflux of thiamin in Escherichia coli; Nishimune T et al.; A resting cell of Escherichia coli lacking thiamin kinase incorporated external thiamin with an energy-dependent counterflow efflux (C-efflux) . This C-efflux could be separated frm an energy-dependent exit by a selective inhibition of exit by 2 x 10(-2) M NaN3 . The extracellular thiamin could be replaced by thiamin diphosphate, resulting in the same rate of C-efflux, but the rate of C-efflux of intracellular thiamin diphosphate against the external thiamin was markedly low . This low rate of C-efflux of thiamin diphosphate could explain the higher accumulation of the compound than that of free thiamin in the thiamin-kinase-defective mutant as well as in its wild-type parent . Basic characteristics of free thiamin uptake and exit in E . coli W mutant were compared with those reported in K 12 mutant: a marked difference existed in the rate of exit . The low rate of exit in E . coli W 70-23-102 was inferred as the reason for the absence of an overshoot phenomenon of thiamin uptake in this strain.

Biochim Biophys Acta, 1981 Dec 7, 649(2), 377 - 84
Inhibition of growth of Escherichia coli by lactose and other galactosides; Wilson DM et al.; A study has been made of the inhibition of growth caused by the addition of lactose or other galactosides to lac constitutive Escherichia coli growing in glycerol minimal medium . The effect was greater at pH 5.9 and pH 7.9 than at pH 7.0 . Inhibition of growth by lactose was observed also in the case of a beta-galactosidase negative mutant . However, a lacY mutant, which has a defect in the entry of protons normally coupled with galactoside transport, showed only slight inhibition of growth on the addition of galactosides . In the case of the parental strain the addition of lactose resulted in a sharp fall in delta pH across the cell membrane and a reduction in intracellular ATP, and the recovery was slow . Under the same conditions the lacY mutant showed a smaller and only transient effect . It is postulated that the sudden entry of protons associated with lactose uptake lowers the protonmotive force, reducing the ATP levels and inhibiting growth of the cells . This hypothesis would account also for the selection of lacY mutants found when E . coli is grown in the presence of isopropyl-beta-D-thiogalactoside.

Vet Rec, 1981 Dec 5, 109(23), 513 - 4
Effect of immunising pregnant sows with different Escherichia coli vaccines on the antibody levels in the piglet sera; Logan EF et al.; Six groups of pregnant sows were immunised with four different commercial Escherichia coli vaccines . Five groups were vaccinated parenterally while the other group was vaccinated orally and then parenterally . The colostral antibody titres to E coli O149 K91(B) K88 as measured by indirect haemagglutination were significantly higher in the vaccinated sows than in the controls . Oral priming by vaccine feeding as opposed to parenteral administration did not appear to produce an anamnestic response . Antibody levels in the piglets born to vaccinated sows were higher than in those born to control sows . Nevertheless a substantial proportion of piglets born to all sows had very low levels of passively acquired immunity.

Biochim Biophys Acta, 1981 Dec 4, 678(2), 165 - 71
In vivo incorporation of selenalysine in Escherichia coli proteins and its effects on cell growth; Cini C et al.; The presence of selenalysine in the culture medium at concentration ranging from 0.05 to 0.3 mM inhibits Escherichia coli growth rate and cell viability . The inhibition of cell growth rate can be imputed to the inhibition of protein synthesis and can be only partially reverted by lysine . Selenalysine is incorporated into cellular proteins in substitution of and in competition with lysine, reaching the value of about 1% as molar fraction with respect to the total amino acids, and substituting up to 14% of protein lysine . The effect of selenalysine on cell viability can be correlated to the extent of its incorporation into proteins, and can be completely reverted by lysine . However, substitution up to 5% of protein lysine by selenalysine does not affect the viability, thus indicating that some degree of substitution can be well tolerated by the cell.

Science, 1981 Dec 4, 214(4525), 1125 - 9
Cloned viral protein vaccine for foot-and-mouth disease: responses in cattle and swine; Kleid DG et al.; A DNA sequence coding for the immunogenic capsid protein VP3 of foot-and-mouth disease virus A12, prepared from the virion RNA, was ligated to a plasmid designed to express a chimeric protein from the Escherichia coli tryptophan promoter-operator system . When Escherichia coli transformed with this plasmid was grown in tryptophan-depleted media, approximately 17 percent of the total cellular protein was found to be an insoluble and stable chimeric protein . The purified chimeric protein competed equally on a molar basis with VP3 for specific antibodies to foot-and-mouth disease virus . When inoculated into six cattle and two swine, this protein elicited high levels of neutralizing antibody and protection against challenge with foot-and-mouth disease virus.

Arch Microbiol, 1981 Dec 2, 130(5), 353 - 6
Iron uptake and iron limited growth of Escherichia coli K-12; Hartmann A et al.; Cells of Escherichia coli K-12 could grow aerobically at an iron concentration as low as 0.05 micrometer without any of the known iron ionophores present . The growth rate increased between 0.05 and 2 micrometer iron . Supplementation with the iron ligands ferrichrome and citrate resulted in optimal growth already at 0.05 micrometer iron . Under certain conditions iron uptake preceded growth of cells by more than an hour . During logarithmic growth the rate of iron uptake matched the growth rate . The radioactive tracer method revealed a cellular iron content of 4 nmol/mg dry weight . After consumption of the iron in the medium cells continued to grow with high rate for 1-2 generations . The iron uptake activity was increased during iron starvation.

Natl Cancer Inst Monogr, 1981 Dec, (58), 139 - 42
Formation of N-2-fluorenylhydroxylamine adducts of DNA in vivo and in vitro and some of their properties; Kriek E et al.; The major 2-fluorenylamine-DNA derivative formed in vivo in rat liver after application of N-2-flourencylacetamide is N-(deoxyguanosin-8-yl)-2-fluorenylamine . This nucleoside, hydrolyzed under mild alkaline conditions with the opening of the imidazole ring, formed two pyrimidine derivatives which can be separated by Sephadex LH-20 column chromatography and thin-layer chromatography on silica . The hydrolysis reaction was catalyzed by metal ions and alkaline phosphatase from Escherichia coli.

Agents Actions, 1981 Dec, 11(6-7), 648 - 50
Platelet thromboxane production during endotoxin shock; Prancan A et al.; Circulating thromboxane (TX) is elevated following endotoxin, and platelets become hyperaggregable . Thromboxane synthesis was therefore studied in platelets during endotoxemia . Rabbit blood and platelets were taken at 0, 60 and 120 min after start of E . coli endotoxin infusion (1.10 microgram/kg min, i.v.) . Blood incubation with arachidonic acid (AA, 10(-4) M) generated TXA2, which was measured using a superfused rabbit aorta bioassay . Washed platelets were stimulated with 1-14C AA (0.1 microCi) to generate radiolabeled TXB2, which was isolated by TLC and quantitated by scintillation spectrometry . Control (0 time) platelet count was 488 +/- 10(3)/mm3 . In the test group, platelet counts fell to 65% of control at 60 min and to 52% at 120 min, while TXA2 generation was 95% (60 min) and 94% (120 min) of control . In contrast a serial dilution of untreated platelets yielded a progressive decline in thromboxane generation . In endotoxemic platelets, the conversion of 1-14C AA to TXB2 (percent/10(9) platelets) was increased at 120 min (0 min, 34.7; 120 min, 40.0: P less than 0.05) . Endotoxemic platelets generated greater amounts of thromboxane than did normal platelets, and this condition may account for platelet hyperaggregability in shock.

J Biochem (Tokyo), 1981 Dec, 90(6), 1705 - 14
A comparison between homologous and heterologous RNA polymerase recognition sites during in vitro chromatin transcription; Tomi H et al.; To analyze the in vitro specificity of homologous and heterologous transcription systems, the template specificities of pea and cauliflower RNA polymerase II for pea chromatin were studied and compared with the specificity exhibited by the Escherichia coli holoenzyme . The results with pea chromatin show that the two eukaryotic enzymes compete with each other for the same recognition sites, but the E . coli enzyme utilizes different recognition sites . The data indicate that during transcription of chromatin in vitro, heterologous eukaryotic RNA polymerase II recognizes the same sites as the homologous enzyme, but the E . coli enzyme does not c enzymes compete with each other for the same recognition sites, but the E . coli enzyme utilizes different recognition sites . The data indicate that during transcription of chromatin in vitro, heterologous eukaryotic RNA polymerase II recognizes the same sites as the homologous enzyme, but the E . coli enzyme does not c enzymes compete with each other for the same recognition sites, but the E . coli enzyme utilizes different recognition sites . The data indicate that during transcription of chromatin in vitro, heterologous eukaryotic RNA polymerase II recognizes the same sites as the homologous enzyme, but the E . coli enzyme does not recognize these sites . In contrast to the situation with chromatin, all three enzymes utilize the same recognition sites on DNA . These results suggest the presence of proteins in chromatin which specifically interact with eukaryotic RNA polymerase II but not with the bacterial enzyme, or a possible template structure in chromatin which can be recognized by eukaryotic enzyme but not by E . coli enzyme.

Infect Immun, 1981 Dec, 34(3), 864 - 70
Inhibition of formylmethionyl-leucyl-phenylalanine-stimulated neutrophil chemiluminescence by human immunoglobulin A paraproteins; Van Epps DE et al.; Immunoglobulin A (IgA) paraproteins from patients with myeloma have been shown to inhibit human neutrophil chemotaxis to C5a, casein, and chemotactic factors produced by Escherichia coli . This study demonstrates that these paraproteins also inhibit neutrophil chemotaxis in response to the synthetic peptide formylmethionyl-leucyl-phenylalanine (f-MLP) . Furthermore, the neutrophil chemiluminescence response stimulated by f-MLP was markedly suppressed by the presence of IgA paraprotein . Maximal inhibition of chemiluminescence was observed when the paraprotein was present during the chemiluminescence response . The inhibitory activity was substantially reduced by removal of the Fc region of IgA or by conversion of polymeric IgA to monomeric IgA by limited reduction and alkylation . Additional experiments showed that these IgA paraproteins inhibited C5a but not phorbol myristate acetate-stimulated chemiluminescence . These observations are constant with the hypothesis that polymeric forms of IgA bind to human neutrophils and interfere with the binding of chemotactic factor to its receptor or the consequent receptor-mediated oxidative burst or both.

Vet Res Commun, 1981 Dec, 5(2), 171 - 5
Studies on Escherichia coli vaccines: estimation of endotoxins in veterinary vaccines; Hussaini SN et al.; Fifteen batches of E . coli vaccines for pigs, from three different manufacturers, were subjected to a quantitative form of the in vitro Limulus-amoebocyte-lysate (LAL) test for their free endotoxin content . A batch of vaccine associated with abortions in pregnant sows was found to contain a much higher level of free endotoxin than batches of vaccine not associated with abortion . The evidence of these assays suggests that they will be useful in the quality control of E . coli vaccines.

J Gen Microbiol, 1981 Dec, 127 (Pt 2), 269 - 76
Physiological behaviour of Escherichia coli grown in opposing gradients of oxidant and reductant in the gradostat; Lovitt RW et al.; Gradients of nutrients are extremely common in nature, and this paper describes changes in the physiology of Escherichia coli grown in the gradostat, a series of five linked vessels with opposing gradients of glucose and of oxygen plus nitrate . Most growth occurred at the aerobic and anaerobic ends of the system . High rates of respiration, high energy charge and high activities of various oxidative enzymes were seen in the two most aerobic vessels; however, oxygen provision was presumably poor, because nitrate reductase activities were also high in this region . Vessels 3 and 4 showed the lowest values for respiration rate, enzyme activity and energy charge, and cells here were both nutrient starved and possibly inhibited by nitrite . Vessel 5 was highly anaerobic, resulting in the presence of hydrogenase activity . It was concluded that cells found in different regions of the gradostat had undergone biochemical differentiation in spatial gradients of electron donors and acceptors.

Gene, 1981 Dec, 16(1-3), 63 - 71
Expression in Escherichia coli of a chemically synthesized gene for a "mini-C" analog of human proinsulin; Wetzel R et al.; A gene has been constructed which codes for an analog of human proinsulin in which the normal 35-amino acid connecting peptide is replaced by a "mini-C" peptide of six amino acids (Arg-Arg-Gly-Ser-Lys-Arg) . The gene, composed of oligonucleotide fragments synthesized by the triester method, was cloned and expressed as a beta-galactosidase hybrid protein . The proinsulin analog was separated from beta-galactosidase by cyanogen bromide cleavage and purified . Controlled disulfide exchange in the S-sulfonate of the analog generated a molecule having high-pressure liquid chromatography (HPLC) and radioimmunoassay (RIA) behavior consistent with a proinsulin-like structure.

Gene, 1981 Dec, 16(1-3), 275 - 86
Construction and characterization of a novel two-plasmid system for accomplishing temperature-regulated, amplified expression of cloned adventitious genes in Escherichia coli; Sninsky JJ et al.; We describe a two-plasmid system that utilizes the lacZ gene promoter and temperature-responsive plasmid replicons to accomplish closely regulated high-level expression of heterologous genes in Escherichia coli . One of the plasmids fails to replicate at 42 degrees C and contains a gene encoding the lac repressor; the second plasmid, which undergoes multicopy "runaway" replication at elevated temperatures, contains an adventitious gene under control of the operator-promoter system of the lacZ gene . Concurrent derepression of lac promoter function and amplification of copy number of the lac-controlled gene occurs when the temperature is elevated . We have used a structural gene encoding chloramphenicol acetyltransferase to demonstrate that the gene product under control of the lacZ promoter represents a major fraction of the total protein synthesized at 43 degrees C, whereas only minimal quantities of this enzyme are made at 30 degrees C . The system described allows the controlled expression of gene products that may have detrimental effects on cell growth, and provides a simple method for identifying radioactivity-labeled protein products of cloned genes in bacterial whole-cell extracts . The system also offers an alternative to intragenic temperature-sensitive mutations for studying the function of various enzymatic or regulatory proteins.

Gene, 1981 Dec, 16(1-3), 27 - 34
Molecular cloning of cDNA sequences coding for the major (beta-, gamma-, delta- and epsilon-) heat-shock polypeptides of HeLa cells; Cato AC et al.; The method of differential in situ colony hybridisation and the technique of two-dimensional gel electrophoresis of polypeptides synthesized in vitro by mRNAs that hybridise specifically to recombinant plasmids bound to filter discs, have been used to isolate two cDNA HeLa heat-shock clones . Each of these two clones contains the plasmid pBR322 with a cDNA fragment of about 0.3 kb inserted into the PstI site . The two fragments hae different restriction maps . Whilst one of the recombinant plasmids, pHS2, contains a cDNA sequence that hybridises to mRNA coding for the HeLa heat-shock gamma-polypeptide, the other, pHS6, cross-hybridises with mRNAs that code for the HeLa heat-shock beta-, delta- and epsilon-polypeptides.

Biochem J, 1981 Dec 1, 199(3), 473 - 7
The effects of anions on fumarate reductase isolated from the cytoplasmic membrane of Escherichia coli; Robinson JJ et al.; A broad range of anions was shown to stimulate the maximal velocity of purified fumarate reductase isolated from the cytoplasmic membrane of Escherichia coli, while leaving the Km for fumarate unaffected . Reducing agents potentiate the effects of anions on the activity, but have no effect by themselves . Thermal stability, conformation as monitored by circular dichroism and susceptibility to the thiol reagent 5,5'-dithiobis-(2-nitrobenzoic acid) are also altered by anions . The apparent Km for succinate in the reverse reaction (succinate dehydrogenase activity) varies as a function of anion concentration, but the maximal velocity is not affected . The membrane-bound activity is not stimulated by anions and its properties closely resemble those of the purified enzyme in the presence of anions . Thus it appears that anions alter the physical and chemical properties of fumarate reductase, so that it more closely resembles the membrane-bound form.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1981 Dec, 251(2), 177 - 84
Chlorhexidine resistance in Escherichia coli isolated from clinical lesions; Nakahara H et al.; Clinical isolates of Escherichia coli (148 strains) were studied for their resistance to chlorhexidine, six antibiotics and three metals . The distribution pattern of their susceptibility to chlorhexidine clearly revealed two peaks, and the resistance was differentiated by 5 micrograms/ml of chlorhexidine . Resistance to chlorhexidine was found in 12.8% of the isolates . The frequencies of resistance to SM, TC, CP, KM, CER, GM, Hg, Cd and As were 43.2, 36.4, 18.9, 25.0, 16.2, 4.1, 35.1, 91.2 and 47.2%, respectively . The frequency of chlorhexidine resistance was lower than that of drug and metal resistance except in the case of GM resistance . Furthermore, all of these chlorhexidine-resistant strains were multiple-drug-resistant also multiple-metal-resistant.

Rev Esp Fisiol, 1981 Dec, 37(4), 463 - 8
Centrifugal field effects on the sedimentation coefficient of the Escherichia coli nucleoid after heat treatment; Pellon JR et al.; The effects of the centrifugal field on the sedimentation coefficient of the heated (50 degrees C, 30 min) Escherichia coli nucleoid were investigated . Form 3,000 r.p.m . the sedimentation coefficients of the heated nucleoids were highly dependent on rotor speed . At 3,000 r.p.m . thier sedimentation coefficient was about 4,000 S while at 7,000 r.p.m . if was about 1,500-1,700 S . At 7,000 r.p.m . and over, nucleoid aggregations occurred and it was difficult to differentiate speed dependence from nucleoid aggregation . Factors likely to cause speed dependence and/or nucleoid aggregation are indicated . The practical importance of these findings is pointed out.

Med Biol, 1981 Dec, 59(5-6), 279 - 85
The complexity of regulation of ornithine decarboxylase; Canellakis ES et al.; Evidence is provided from Escherichia coli, from mammalian cells as well as from germinating barley seeds that there exist positive and negative macromolecular effectors of ODC (ornithine decarboxylase) that modify its activity . These effectors interact with ODC either by forming inactive complexes or by lowering its KmORN resulting in the activation of ODC . These facts, in addition to other evidence presented, argue for the existence of an equilibrium between: (Formula: see text) Such an equilibrium would result in the modulation of ODC activity independently of the net synthesis or degradation of ODC molecules.

Proc Natl Acad Sci U S A, 1981 Dec, 78(12), 7483 - 6
Enzymatic reduction of oxidized alpha-1-proteinase inhibitor restores biological activity; Abrams WR et al.; The major serum inhibitor of proteolytic activity, alpha-1-proteinase inhibitor (alpha-1-PI), (or alpha-1-antitrypsin) can be readily inactivated by oxidation {Carp, H . & Janoff, A . (1978) Am . Rev . Resp . Dis . 118, 617-621} . This inactivation appears to be due to the oxidation of a critical methionine(s) in alpha-1-PI that is required for the inhibition of elastase activity . An enzyme from Escherichia coli that reduces methionine sulfoxide residues in protein {Brot, N., Weissbach, L., Werth, J . & Weissbach, H . (1981) Proc . Natl . Acad . Sci . USA 78, 2155-2158} can restore the biological inhibitory activity of canine oxidized alpha-1-PI.

Proc Natl Acad Sci U S A, 1981 Dec, 78(12), 7408 - 11
Nucleotide insertion in the anticodon loop of a glycine transfer RNA causes missense suppression; Prather NE et al.; We have determined the nucleotide sequences of two unusual UGG-suppressing glycine tRNAs from Escherichia coli and, as a result, have discovered a new mechanism for the generation of missense suppressors . The suppressor tRNAs translate UGG but not UGA . Each arose as a consequence of spontaneous mutational alteration of glyT, the gene for the GGA/G-reading glycine tRNA of E . coli . In each mutant tRNA, the change in primary structure involved the insertion of an adenylate residue on the 3' side of the anticodon and the loss of a modification of the uridylate residue at the 5' end of the anticodon . A "shift" of the effective anticodon by one nucleotide in the 3' direction can account for the new coding specificity of these tRNAs.

Proc Natl Acad Sci U S A, 1981 Dec, 78(12), 7261 - 5
Yeast 2-micrometer plasmid DNA replication in vitro: origin and direction; Kojo H et al.; Most yeast strains harbor extrachromosomal 2-micrometer DNA, and this DNA synthesis, like nuclear DNA replication, is strictly under cell cycle control . A soluble extract of yeast Saccharomyces cerevisiae carries out semiconservative replication of added 2-micrometer DNA and Escherichia coli chimeric plasmids containing the 2-micrometer DNA . Replication is initiated on 10% of the DNA, and one round of replication is completed . The major products in early stages of replication are theta ("eye") forms which originate 140 +/- 50 nucleotides within one of the 599-base-pair inverted repeats of 2-micrometer DNA . Their replication is bidirectional and discontinuous . Extracts prepared from the cell division cycle mutant cdc8 show temperature-sensitive 2-micrometer DNA synthesis in vitro, suggesting that this in vitro system resembles in vivo 2-micrometer plasmid DNA replication . This system should provide a useful assay for the purification and characterization of yeast DNA replication proteins.

Mutat Res, 1981 Dec, 84(2), 257 - 62
Effect of the uvrD3 mutation on ultraviolet radiation-induced DNA-repair replication in Escherichia coli K12; Carlson KM et al.; Ultraviolet-radiation-induced DNA-repair replication was measured in wild-type, polA1, uvrD3, and polA1 uvrD3 strains of Escherichia coli K12 . A large stimulation of repair replication was observed in the uvrD3 strain, compared to the wild-type and polA1 strains . This enhanced repair replication was reduced in the polA1 uvrD3 strain . Therefore, uvrD3 mutation appears to affect the amount of repair replication performed by DNA polymerase I . In the polA1 strain, there also appears to be an effect of the uvrD3 mutation on the amount of repair replication performed by DNA polymerase III (and/or II) . The enhanced repair replication observed for the uvrD3 strains appears to be in response to the enhanced DNA degradation observed for these strains.

Mutat Res, 1981 Dec, 84(2), 227 - 37
Mutation induction by monochromatic 254-nm and 365-nm radiation in strains of Escherichia coli that differ in repair capability; Webb RB et al.; Mutation to tryptophan independence after exposure to radiation at the monochromatic wavelengths of 254 and 365 nm was studied and compared in 7 strains of Escherichia coli B/r that differ in repair capability . Efficient mutation induction was obtained with both 254-nm and 365-nm radiation with strains WP2 (wild-type), WP2s (uvrA), WP6s (polA), and WP6 (polA uvrA) . Mutants were not induced at either wavelength in the lexA strain WP5 or the recA strains WP10 and WP100 . These results support the induction of mutants with 365-nm radiation through the error-prone (SOS) pathway of postreplication repair . Log-log plots of tryptophan revertant data at 254 nm showed the expected slopes of approximately 2.0 over the entire fluence range tested . In contrast, similar plots of revertant data at 365 nm were complex in all cases tested: at low fluence values (survival greater than 0.5) in all cases where reversion occurred the slopes were approximately 1.0, while at higher fluences (survival less than 0.5) the slopes of the log-log plots were approximately 3.0 with strains WP2s and WP6s, approximately 4.0 with strain WP6, and approximately 6.0 with strain WP2 . Differential sensitivity of components of excision and postreplication repair systems to 365-nm radiation may account for the 2-part mutation curves obtained with uvr+ rec+ lex+ strains . It is proposed that efficient error-free repair of mutational lesions occurs at 365-nm fluences below 2-4 x 10(5) J m-2; at greater 365-nm fluences, error-free excision repair may be selectively inhibited, forcing a greater fraction of mutational lesions to be processed by the error-prone component of the postreplication repair system . The similarity of the mutational responses of WP2s and WP6 at 365 nm supports the selective inhibition of error-free excision repair.

J Radiol, 1981 Dec, 62(12), 629 - 33
{Extramucosal parietal lesions of the sigmoid of different aetiologies in children: report on two cases (author's transl)}; Bretagne MC et al.; Rare, atypical, extramucosal parietal lesions in the rectosigmoid regions were observed in two children . The first child, with Henoch-Schonlein purpura, developed an intramural haematoma, a complication rarely reported in the literature . The second child had a perivisceral abscess of probable adnexial origin, arising during the course of pelvic septicaemia.

Eur J Biochem, 1981 Dec, 120(3), 497 - 509
Origin and characterization of short DNA chains in Escherichia coli; Siegmann DW et al.; One of the major problems in the study of DNA replications involves the presence of numerous short, non-nascent DNA chains in the cell . Such chains often contaminate nascent DNA preparations, making accurate analysis of the replicative DNA difficult . This complication can be avoided by the use of hydroxyapatite chromatography . Previous results from this laboratory had shown that short nascent DNA chains could be eluted from hydroxyapatite at low phosphate concentrations because of their non-covalent association with protein . Additional data now indicate that this isolation procedure yields a DNA preparation which contains only nascent DNA and is essentially free of non-nascent chains . 5'-Terminal labelling patterns and molecular weight distributions show that only nascent DNA chains from the DNA-protein complex and can therefore be separated from non-nascent chains if the isolation is performed under non-denaturing conditions . The protein involved in the complex appears to be rather specific for DNA chains in the replication fork since short chains resulting from the excision of dUMP immediately after replication also form the DNA-protein complex . Using this isolation technique, the 5'-terminal nucleotides of nascent DNA chains were determined in an effort to learn something about the chain initiation process . DNA was pulse-labeled in several ways, purified by hydroxyapatite chromatography, and labeled at the 5' end with {32P}phosphate . Two-dimensional thin-layer chromatography of the 32P-labeled nucleotides, obtained after enzymatic hydrolysis, revealed that all four deoxyribonucleotides are present at the 5' end in approximately equal amounts . In most cases, this 5'-terminal nucleotide distribution does not vary significantly, even when the conditions of pulse-labeling are changed . Only if the cells are allowed to run out of thymine is a variation in the relative amounts of 5'-end nucleotides detected . In this case, the amount of dTMP at the 5' end decreases significantly, reflecting the low thymine levels and indicating that the cell can compensate for this deficiency by utilizing other available nucleotides . From these results, it appears that DNA chain initiation is essentially random with respect to the nucleotide used in the initiation process.

Can J Microbiol, 1981 Dec, 27(12), 1283 - 9
Effect of carbon source on growth rate and phospholipid composition of Escherichia coli 15T- and an unsaturated fatty acid auxotroph; Urban JE et al.; Escherichia coli 15T- was grown with glucose, succinic acid, aspartic acid, oleic acid, and oleic plus aspartic acids as carbon sources, and a fatty acid auxotroph derived from 15T- was grown on oleic acid and oleic plus aspartic acids . The doubling time, phospholipid composition, phosphorus content, and the fatty acid composition of the phospholipids of cells in each of the media were determined . In all cases, phosphatidylethanolamine was the major phospholipid present; but with 15T- its concentration was inversely proportional to the doubling time in unsupplemented media . With the auxotroph the phosphatidylethanolamine concentration was essentially unchanged with growth . Total lipid phosphorus was inversely proportional to doubling time, an effect particularly evident with the auxotroph . Without oleic acid supplementation, the major effects of carbon source on fatty acid composition are decreases in the content of palmitoleic acid and increases in the content of cis-9, 10-methylene hexadecanoic acid as growth rate decreases . Oleic acid supplementation elevated 18:1 fatty acid content in both 15T- and the auxotroph.

Biokhimiia, 1981 Dec, 46(12), 2160 - 3
{DNA methylation in the cells of Escherichia coli MRE 600 in the presence of S-methylmethionine}; Lebenka AIu et al.; The effect of S-methylmethionine (SMM), a methyl group donor, on enzymatic methylation of DNA in E . coli MRE 600 cells was studied . It was found that SMM can be used as a donor of methyl groups during bacterial DNA methylation in vivo without changing the specificity of DNA methylation.

Urology, 1981 Dec, 18(6), 535 - 41
Diagnostic strategy in evaluation of renal abscess; Godec CJ et al.; The diagnosis of renal abscess is still a challenging problem . Early manifestations are usually nonspecific and nonlocalizing . Clinical picture, laboratory data, and intravenous pyelogram do not always differentiate between inflammatory lesions and neoplastic processes . Two cases with bilateral, metachronous renal abscesses are presented . The use of 111Indium scan is probably the major recent advance used for unmasking this inflammatory lesion . Sonography, computerized tomography scan, and even arteriography are sometimes necessary to establish reliable preoperative diagnosis . The diagnostic strategy for a systematic approach to the lesion is outlined.

J Hyg (Lond), 1981 Dec, 87(3), 413 - 9
Escherichia coli in gastroenteritis of children in Auckland, New Zealand; Goldwater PN et al.; A study of stool Escherichia coli in 60 children with gastroenteritis and 18 control children was carried out in Auckland, New Zealand in 1977 . Toxigenic strains, heat labile and heat stable, predominated in the stools of only three children, all of whom had concomitant rotavirus . Classical enteropathogenic E . coli (EPEC) were found in patients and controls . Only one patient had many EPEC in the stool (086.H2), they were variably toxigenic and rotavirus was also present . No toxigenic serotype was isolated . Two potential pathogens were sometimes found . Overall there was no evidence for a substantial causative role for disease producing E . coli in these children.

J Bacteriol, 1981 Dec, 148(3), 983 - 7
Molecular organization of heat-labile enterotoxin genes originating in Escherichia coli of human origin and construction of heat-labile toxoid-producing strains; Yamamoto T et al.; Recombinant deoxyribonucleic acid technology was employed to construct heat-labile enterotoxin (LT) toxoids . A recombinant plasmid carrying both an LT promoter region and LT subunit A (LTA) gene, lacking as much as 0.25 kilobases of the region up to the C terminus, produced a peptide possessing immunological properties of LTA but lacking the ability to construct LT activity (designated as LTA*) . A cloned LT subunit B (LTB) gene produced LTB when a promoter on a vector was available for the gene . Escherichia coli producing LTA* and LTB (LT toxoids) could be useful as a vaccine.

Proc Natl Acad Sci U S A, 1981 Dec, 78(12), 7478 - 82
Thioredoxin, glutaredoxin, and thioredoxin reductase from cultured HeLa cells; Tsang ML et al.; Thioredoxin and glutaredoxin may be important in regulating cell metabolism by mediating interchanges between sulfhydryl and disulfide groups . Components of the thioredoxin/glutaredoxin system from cultured HeLa cells have been partially purified and characterized by using Escherichia coli adenosine 3'-phosphate 5'-phosphosulfate reductase, a thioredoxin/glutaredoxin-dependent enzyme on the pathway of sulfate reduction, as an assay system . In HeLa cells, a NADPH-thioredoxin reductase and three heat-labile proteins (designated PI, PII, and PIII) that have thioredoxin- or glutaredoxin-like properties are found . Both PI and PIII have molecular masses of approximately 12,000 daltons and are readily reduced by their homologous HeLa thioredoxin reductase . However, only PI can be reduced efficiently by the glutathione system and neither PI nor PIII has inherent glutathione-disulfide oxidoreductase activity . PII has a molecular mass of greater than 30,000 daltons and appears to be associated with a reductase activity . The HeLa NADPH-thioredoxin reductase has been purified to near homogeneity and found to be a 116,000-dalton flavoprotein composed of two 58,000-dalton subunits . The HeLa enzyme has low species and substrate specificity and can reduce HeLa PI and PIII, E . coli thioredoxin and glutaredoxin, and the disulfide bond in 5,5'-dithiobis(2-nitrobenzoic acid) . The exact in vivo roles of the HeLa thioredoxin/glutaredoxin system remain to be determined.

Proc Natl Acad Sci U S A, 1981 Dec, 78(12), 7365 - 9
Nucleotide sequence of cloned cDNAs encoding human preproparathyroid hormone; Hendy GN et al.; We have cloned cDNA copies of human preproparathyroid hormone in Escherichia coli after insertion of double-stranded DNA into the Pst I site of plasmid pBR322 using the poly(dG) . poly(dC) homopolymer extension technique . Recombinant plasmids coding for preproparathyroid hormone were identified by filter hybridization assay using as a probe 32P-labeled bovine preproparathyroid cDNA . Nucleotide sequence analysis of five recombinant plasmids permitted the assignment of 74 nucleotides of the 5' noncoding region, the entire coding region of 345 nucleotides, and the entire 3' noncoding region of 348 nucleotides of the mRNA . The coding sequence predicts the previously unknown "pre" amino acid sequence and clarifies the hormone's amino acid sequence, which has been disrupted . The 5' noncoding region contains an AUG codon followed by a UGA stop codon before the authentic initiator codon . The 3' noncoding region is 120 nucleotides longer than in bovine preproparathyroid mRNA and contains two A-A-U-A-A-A sequences, potential signals for polyadenylation.

Biochem J, 1981 Dec 1, 199(3), 733 - 40
Limited proteolysis and proton n.m.r . spectroscopy of the 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli; Perham RN et al.; The 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli was treated with trypsin at pH 7.0 at 0 degrees C . Loss of the overall catalytic activity was accompanied by rapid cleavage of the lipoate succinyltransferase polypeptide chains, this apparent Mr falling from 50 000 to 36 000 as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis . A slower shortening of the 2-oxoglutarate decarboxylase chains was also observed, whereas the lipoamide dehydrogenase chains were unaffected . The inactive trypsin-treated enzyme had lost the lipoic acid-containing regions of the lipoate succinyltransferase polypeptide chains, yet remained a highly assembled structure, as judged by gel filtration and electron microscopy . The lipoic acid-containing regions are therefore likely to be physically exposed in the complex, protruding from the structural core formed by the lipoate succinyltransferase component between the subunits of the other component enzymes . Proton nuclear magnetic resonance spectroscopy of the 2-oxoglutarate dehydrogenase complex revealed the existence of substantial regions of polypeptide chain with remarkable intramolecular mobility, most of which were retained after removal of the lipoic acid-containing regions by treatment of the complex with trypsin . By analogy with the comparably mobile regions of the pyruvate dehydrogenase complex of E . coli, it is likely that the highly mobile regions of polypeptide chain in the 2-oxoglutarate complex are in the lipoate succinyltransferase component and encompass the lipoyl-lysine residues . It is clear, however, that the mobility of this polypeptide chain is not restricted to the immediate vicinity of these residues.

Biochem J, 1981 Dec 1, 199(3), 505 - 11
The role of lipoic acid residues in the pyruvate dehydrogenase multienzyme complex of Escherichia coli; Danson MJ et al.; Two lipoic acid residues on each dihydrolipoamide acetyltransferase (E2) chain of the pyruvate dehydrogenase multienzyme complex of Escherichia coli were found to undergo oxidoreduction reactions with NAD+ catalysed by the lipoamide dehydrogenase component . It was observed that: (a) 2 mol of reagent/mol of E2 chain was incorporated when the complex was incubated with N-ethylmaleimide in the presence of acetyl-SCoA and NADH; (b) 4 mol of reagent/mol of E2 chain was incorporated when the complex was incubated with N-ethylmaleimide in the presence of NADH; (c) between 1 and 2 mol of acetyl groups/mol of E2 chain was incorporated when the complex was incubated with acetyl-SCoA plus NADH; (d) 2 mol of acetyl groups/mol of E2 chain was incorporated when the complex was incubated with pyruvate either before or after many catalytic turnovers through the overall reaction . There was no evidence to support the view that only half of the dihydrolipoic acid residues can be reoxidized by NAD+ . However, chemical modification of lipoic acid residues with N-ethylmaleimide was shown to proceed faster than the accompanying loss of enzymic activity under all conditions tested, which indicates that not all the lipoyl groups are essential for activity . The most likely explanation for this result is an enzymic mechanism in which one lipoic acid residue can take over the function of another.

Zentralbl Bakteriol Mikrobiol Hyg {B}, 1981 Dec, 174(4), 348 - 54
Sensitivity of various spiroplasma strains against ethanol, formalin, glutaraldehyde, and phenol; Stanek G et al.; The efficacy of four different disinfectants on spiroplasmas pathogenic for plants, insects and vertebrates was determined using a microtiter technique . The results of the sensitivity testing indicate that spiroplasmas display a considerable resistance in comparison to cell wall bearing organisms . Particularly honey bee spiroplasmas proved to be less sensitive to the disinfectants tested, whereas Spiroplasma citri and the tick spiroplasmas showed a susceptibility comparable to E . coli and Staph . aureus . The findings are discussed in relation to the technique used and in respect of the implications for laboratory work.

Proc Natl Acad Sci U S A, 1981 Dec, 78(12), 7336 - 40
Plasmid-directed synthesis of enzymes required for D-mannitol transport and utilization in Escherichia coli; Lee CA et al.; A transformant Escherichia coli colony bank {Clarke, L . & Carbon, J . (1976) Cell 9, 91-99} has been screened for hybrid ColE1 plasmids carrying the genes for D-mannitol utilization . Two of the plasmids, pLC11-7 and pLC15-48, were shown to contain the mannitol operon, which includes the structural genes for the mannitol-specific enzyme II of the phosphotransferase system and mannitol-1-phosphate dehydrogenase . One E . coli strain harboring plasmid pLC15-48 overproduced mannitol-1-phosphate dehydrogenase activity 4- to 5-fold . However, there was no corresponding increase in mannitol enzyme II activity . Plasmid pLC15-48 was shown to direct the synthesis of two polypeptides in E . coli minicells in the presence of cyclic AMP and mannitol . The larger (Mr = 60,000) was membrane bound and was specifically precipitated by antibody directed against purified mannitol-specific enzyme II . The smaller (Mr = 40,000) was soluble and had an electrophoretic mobility indistinguishable from that of the major component in a partially purified mannitol-1-phosphate dehydrogenase preparation . These data are consistent with previous genetic studies of the mannitol locus and confirm an independent conclusion {Jacobson, G . R., Lee, C . A . & Saier, M . H., Jr . (1979) J . Biol . Chem . 254, 249-252} that mannitol enzyme II consists of a single type of polypeptide chain that has a Mr of 60,000 . The plasmid pLC15-48 DNA was characterized by mapping of restriction endonuclease cleavage sites.

Orig Life, 1981 Dec, 11(4), 343 - 51
Origins of translation: the hypothesis of permanently attached adaptors; Tyagi S; A mechanism for prebiotic translation is proposed in which primeval transfer-RNA (adaptors) are assumed to be permanently associated with messenger nucleic acid molecules . Residual 'fossil' evidences are found to be present within the base sequences of contemporary tRNAs, suggesting the existence of inter-primal-tRNA interactions necessary for the mechanism . The structure of proposed primal-tRNA is such that it can not only choose its own amino acid in the absence of aminoacyl synthetase, but can also associate nonspecifically with adjacent primal-tRNA molecules attached to the neighbouring codons . Such associations can give rise, through cooperative binding between message and adaptors to the 'static template surfaces' which can direct translation of nucleotide sequences into those of amino acids . The origins of ribosomes and contemporary genetic code are suggested by this hypothesis . Proposed structures and processes are thermodynamically compatible . The approximate date of occurrence of the proposed system is calculated, which is consistent with the period of occurrence of the earliest organism with ribosomes.

Hoppe Seylers Z Physiol Chem, 1981 Dec, 362(12), 1567 - 74
Dependency on serine concentration of the activity of tryptophan synthase . Cooperative properties; Heilmann HD et al.; The activity of the enzyme tryptophan synthase from Escherichia coli was tested as a function of the concentration of L-serine which serves as a substrate in the indole to tryptophan reaction as well as for the L-serine deaminase activity . L-Serine binding was found to follow the pattern of negative cooperativity both by kinetic and by equilibrium methods . The enzyme kinetic data support the view that a rapid equilibration model for the enzyme . substrates complex formation is not strictly obeyed.

J Bacteriol, 1981 Dec, 148(3), 926 - 32
Regulation of coenzyme A biosynthesis; Jackowski S et al.; Coenzyme A (CoA) and acyl carrier protein are two cofactors in fatty acid metabolism, and both possess a 4'-phosphopantetheine moiety that is metabolically derived from the vitamin pantothenate . We studied the regulation of the metabolic pathway that gives rise to these two cofactors in an Escherichia coli beta-alanine auxotroph, strain SJ16 . Identification and quantitation of the intracellular and extracellular beta-alanine-derived metabolites from cells grown on increasing beta-alanine concentrations were performed . The intracellular content of acyl carrier protein was relatively insensitive to beta-alanine input, whereas the CoA content increased as a function of external beta-alanine concentration, reaching a maximum at 8 microM beta-alanine . Further increase in the beta-alanine concentration led to the excretion of pantothenate into the medium . Comparing the amount of pantothenate found outside the cell to the level of intracellular metabolites demonstrates that E . coli is capable of producing 15-fold more pantoic acid than is required to maintain the intracellular CoA content . Therefore, the supply of pantoic acid is not a limiting factor in CoA biosynthesis . Wild-type cells also excreted pantothenate into the medium, showing that the beta-alanine supply is also not rate limiting in CoA biogenesis . Taken together, the results point to pantothenate kinase as the primary enzymatic step that regulates the CoA content of E . coli.

J Pharmacol Exp Ther, 1981 Dec, 219(3), 778 - 85
Hypothermia induced in rabbits by intracerebroventricular taurine: specificity and relationships with central serotonin (5-HT) systems; Sgaragli G et al.; The intracerebroventricular (i.c.v.) injection of taurine produced a fall in core temperature, the extent of which was dependent on the thermal gradient between the body and the environment . Concurrently, a sudden rise in ear skin temperature, which was maximal in the cold and negligible at 30 degrees C, was observed . The fever induced by i.v . injection of Escherichia coli endotoxin was antagonized by taurine . High temperatures produced by i.c.v . injection of prostaglandin E1 were also suppressed by taurine . Intracerebroventricular injections of bicuculline and strychnine, but not those of picrotoxin or pentylentetrazol, were able to reduce hypothermia induced by taurine . Intracerebroventricular injection of the taurine reuptake inhibitor guanidinoethyl sulfonate, on the contrary, did enhance the hypothermic response to taurine . Injection (i.c.v.) of serotonin (5-HT) elicited a fall in core temperature which was not accompanied by a rise in ear skin temperature, but was antagonized by the concurrent injection of the 5-HT antagonist methysergide . Pretreating animals with p-chlorophenyl-alanine caused a significant fall of brain 5-HT contents and a reduction of the hypothermic response to taurine . The latter effect was also observed when the animals were i.c.v . pretreated either the methysergide or with the 5-HT reuptake blockers chlorimipramine and Lilly 110140 . These findings give support to the hypothesis that taurine-induced hypothermia in rabbits mediated by some taurine sensitive cells and, at least in part, by serotonergic synaptic mechanisms.

Gene, 1981 Dec, 16(1-3), 287 - 95
Functional expression of the Herpes simplex virus thymidine kinase gene in Escherichia coli K-12; Kit S et al.; The recombinant plasmid pAGO contains the Herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene and consists of a 2-kb PvuII fragment of HSV-1 DNA inserted into the PvuII site of pBR322 . A deletion mutant of pAGO, designated pMH110, has been isolated which removes the normal HSV-1 TK gene promoter but places the promoter of the pBR322 tetracycline-resistance (tetr) gene only about 400 bp from the translational start codon of the HSV-1 TK polypeptide . In contrast to pAGO, which transforms mouse LM(TK-) cells to TK+ but is only weakly expressed in TK- bacteria, pMH110 not only efficiently transforms LM(TK-) cells to TK+ but also enables TK- Escherichia coli K-12 cells to form colonies on selective plates containing 5-fluorodeoxyuridine (FdUrd) plus thymidine (dThd) and to exhibit fully restored ability to incorporate {3H}dThd into DNA . The levels of TK activity expressed by bacteria harboring pMH110 were about as high as those expressed by bacteria harboring plasmid pTK3, which contains the wild-type E . coli TK gene . The TK activity expressed in bacteria harboring pMH110 was partially purified and shown to be HSV-1-specific by serological and disc PAGE analyses and by experiments demonstrating that this enzyme phosphorylated {125I}deoxycytidine.

Gene, 1981 Dec, 16(1-3), 217 - 25
An avian tumor virus promoter directs expression of plasmid genes in Escherichia coli; Mitsialis SA et al.; A sequence in the long terminal repeat (LTR) of avian tumor virus (ATV) DNA was shown to contain a promoter acute in Escherichia coli . For this analysis the bacterial promoters for the tetracycline (Tc) and neomycin (Nm) resistance genes were deleted from different plasmids and replaced with various fragments derived from the ATV DNA . Expression of the drug-resistant phenotype in the recombinant plasmids at levels comparable to or greater than those found with parental bacterial promotes was shown to be dependent on the presence of an intact sequence ranging from nucleotide +19 to -23 (relative to the cap site) in the ATV DNA . Comparison of the consensus bacterial promoter with the nucleotide sequence in this region revealed strong similarities.

Gene, 1981 Dec, 16(1-3), 21 - 6
A set of synthetic oligodeoxyribonucleotide primers for DNA sequencing in the plasmid vector pBR322; Wallace RB et al.; Seven oligonucleotide primers complementary to the plasmid vector pBR322 at positions adjacent to five of the unique restriction endonuclease cleavage sites (EcoRI, HindIII, BamHI, SalI and PstI) have been chemically synthesized . The polarity of the primers is such that any DNA inserted at one or a combination of two of the above restriction sites may be sequenced by the chain termination method using one of the synthetic DNA primers . One of the primers for sequencing inserts at the PstI site of pBR322 is also complementary to the M13 phage vector designated bla6 . This set of universal primers is useful for rapid sequence determination of DNA cloned into pBR322 or M13bla6.

Gene, 1981 Dec, 16(1-3), 207 - 16
Cloning of the human cytomegalovirus genome as endonuclease XbaI fragments; Thomsen DR et al.; Restriction enzyme XbaI DNA fragments that represent 99% of the sequences from the long and short unique as well as the repeat sequences of the human cytomegalovirus (CMV) genome have been cloned into bacterial plasmid pACYC184 . The viral DNA sequences associated with the recombinant plasmids were analyzed by restriction mapping and by hybridization to fragments of authentic viral DNA . The relationship of the cloned viral DNA fragments to the XbaI physical map of the viral genome is demonstrated . Even though large recombinant plasmids ranging from approx . 39 to 1.8 kb were isolated, most if not all of the viral DNA fragments were stable during propagation in Escherichia coli HB101.

Gene, 1981 Dec, 16(1-3), 191 - 7
Construction and characterization of three yeast-Escherichia coli shuttle vectors designed for rapid subcloning of yeast genes on small DNA fragments; Ferguson J et al.; We have constructed three new subcloning plasmid vectors, pRC1, pRC2, and pRC3, derived from pKC7, which allow the rapid, single-step subcloning of yeast genes . Subcloning with these vectors utilizes a partial digestion with Sau3A to generate a quasi-random set of DNA fragments from the original plasmid . All three vectors contain a kanamycin resistance gene . Therefore, if the original cloned yeast DNA fragment is present in a vector that does not specify kanamycin resistance, the subclone pool can be propagated in Escherichia coli in the presence of kanamycin to select against parent plasmids that escaped restriction by Sau3A . Selection by complementation in yeast yields a collection of plasmids with smaller yeast DNA inserts containing the gene of interest . In the vectors pRC2 and pRC3, constructed from pRC1, the unique BamHI site is located within an intact tetracycline resistance gene, thus making it possible to screen bacterial transformants for those containing recombinant plasmid molecules . Vectors pRC2 and pRC3 also contain the yeast 2 micrometers DNA replication origin, and thus are more stable than plasmids carrying only the TRP1-associated replicator (ars1).

Gene, 1981 Dec, 16(1-3), 171 - 7
Solution hybridization using cloned, 5'-32P-labeled, double-stranded DNA to distinguish among closely related nucleic acids; Ross J et al.; A rapid, sensitive, and specific solution-hybridization method is described that utilizes cloned, double-stranded DNA . The DNA is treated with restriction enzyme(s), and fragments are 32P-labeled at their 5' termini with polynucleotide kinase . A single fragment is partially purified by gel electrophoresis, denatured, and annealed with unlabeled RNA or DNA . The reaction mix is treated with S1 nuclease, precipitated with TCA, and the {32P}DNA counted . Hybridization is recognized only when the 32P label is associated with duplexes that are TCA-precipitable . The specificity of the method was analyzed by annealing experiments with cloned, human globin DNAs . Restriction fragments labeled in the 3' untranslated region of human beta globin clones formed S1-resistant, TCA-precipitable duplexes with beta globin DNA, but not with delta or gamma globin DNA . Thus, a major advantage of the method is that it can distinguish among homologous nucleic acids whose uniformly labeled cDNAs cross hybridize under moderately stringent conditions . This assay is as sensitive as the {3H}cDNA hybridization method, and it circumvents the requirement for purified mRNA as a template for {3H}cDNA synthesis . It also avoids a gel electrophoresis step, it is rapid and quantitative, and many samples can be simultaneously analyzed.

Gene, 1981 Dec, 16(1-3), 161 - 70
Comparison of IS1, IS2 and IS3 copy number in Escherichia coli strains K-12, B and C; Hu M et al.; The number of copies of IS2 and IS1 in the chromosomes of five Escherichia coli K-12 strains and E . coli B and E . coli C has been determined by hybridization . Among these strains, IS1 copy numbers range from 4 to 19 and IS2 copy numbers range from 0 to 12 . IS2 is present once in the E . coli B chromosome, but it is absent from E . coli C . The copy numbers of IS3 in the same seven E . coli strains range from 4 to 6.

Gene, 1981 Dec, 16(1-3), 119 - 32
Nucleotide sequence of the argF regulatory region of Escherichia coli K-12; Moore SK et al.; The deoxyribonucleotide sequence has been determined for the regulatory region of the arginine F gene (argF) of Escherichia coli K-12 . The location of the argF coding region was deduced by comparison of the DNA sequence to the sequence predicted from the primary structure of the N-terminus of the argF gene product, the subunit of the "F" isoenzyme of ornithine transcarbamylase . Transcription of the argF gene was found to initiate at a position approx . 40 bp preceding the N-terminal codon for OTCase . Comparison of the region surrounding the origin of transcription with a computer-generated "model promoter sequence" revealed structural similarities between the two sequences, in particular, the promoter-associated stretches known as the "Pribnow box" and "minus 35 contact site" . Another feature noted for the argF promoter region was its extreme abundance of A : T nucleotide pairs . In the region preceding the start site for argF translation, a sequence was observed to be complementary to the 3' end of the 16S RNA component of the E . coli ribosome . Both the length and the nucleotide sequence of the argF leader region indicate that the argF gene does not contain an attenuator proposed to exist in other operons concerned with amino acid biosynthesis.

Gene, 1981 Dec, 16(1-3), 1 - 9
Structure and variation of human ribosomal DNA: molecular analysis of cloned fragments; Erickson JM et al.; Eco-RI-A fragments of the human ribosomal RNA gene family from two types of tissue and three individuals were cloned in lambda vectors and compared by restriction enzyme digestion and electron microscopy . The EcoRI fragment A contains (i) 0.2 kb of the 3' end of the 18S rDNA, (ii) 2.5 kb of internal transcribed spacer and the 5.8S rDNA, and (iii) 4.6 kb of the 28S rDNA gene . All of the six cloned rDNA fragments isolated are identical by these analyses . Moreover, all contain a HincII site that is absent in about 50% of the rDNA identified by genomic blotting . Polymorphism in the nontranscribed spacer rDNA was studied in genomic blots of BamHI-digested DNA, using the 3' end of the 28S rDNA as a probe . The boundaries between the 18S rDNA, internal transcribed spacer, 28s rDNA, and external nontranscribed spacer were determined by R-loop analysis, further defining the organization of the ribosomal RNA precursor.

Natl Cancer Inst Monogr, 1981 Dec, (58), 189 - 92
Enzymatic mechanisms of DNA repair; Grossman L; DNA repair proficiency in cells is expressed by various enzymes which can recognize damaged sites arising from exogenous agents or endogenous conditions . Either a damaged base is recognized by DNA glycosylases, partially removed by hemi-DNA glycosylases acting on diadduct damage, or direct incision of the phosphodiester bond near the damaged site . Incision at those apurinic or apyrimidinic sites arising from depurination-depyrimidination or glycosylase reactions is effected by apurinic or apyrimidinic endonucleases . Excision of damaged sites is catalyzed by unique exonucleases followed by DNA polymerase catalyzed reinsertion of nucleotides . The integrity of the strands is restored by polynucleotide ligase when a juxtaposed nucleotide is properly reinserted.

Med Biol, 1981 Dec, 59(5-6), 374 - 80
Effect of polyamines on enzymes involved in DNA repair; Kleppe K et al.; The influence of polyamines on various enzymes involved in the excision repair pathway of DNA, such as UV endonuclease, DNA polymerase I, DNA ligase and polynucleotide kinase, and two AP-endonucleases, were studied . The polymerizing activities of DNA polymerase I and polynucleotide kinase were found to be markedly affected by polyamines . In the former enzyme the effect can be attributed to the stabilization of the correct bihelical structure at the 3' end and in the latter case polyamines stabilize the polynucleotide kinase protein itself in the correct oligomeric structure . The effect of polyamines on the hydrolysis of apurinic and apyrimidinic sites in DNA and nucleosome particles were also investigated . Spermine and spermidine were found to be the most efficient polyamines in causing such hydrolysis both in the free DNA and in the nucleosome particles.

Proc Natl Acad Sci U S A, 1981 Dec, 78(12), 7652 - 6
Mutations in the lacY gene of Escherichia coli define functional organization of lactose permease; Mieschendahl M et al.; Mutations in the lacY gene of Escherichia coli have been used to analyze the functional organization of lactose permease . Deletions suggest that the NH2 terminus of lactose permease is not essential and can be replaced by residues of the cytoplasmic enzyme beta-galactosidase . Negative dominant mutations in the lacY gene can be explained by the assumption that membrane-associated lactose permease is active as a dimer or oligomer . The map positions of these mutations and other point mutations that lower or alter the sugar specificity define regions of lactose permease involved in sugar or proton binding and transport.

Proc Natl Acad Sci U S A, 1981 Dec, 78(12), 7370 - 4
Enzymatic replication of the origin of the Escherichia coli chromosome; Fuller RS et al.; An enzyme system that replicates plasmids bearing the origin of the Escherichia coli chromosomes (oriC) has the following physiologically relevant features . The system (i) depends completely on low levels of exogenously furnished supercoiled oriC plasmids, (ii) uses only those plasmids that contain the intact oriC region of about 245 base pairs, (iii) initiates replication within or near the oriC sequence and proceeds bidirectionally, (iv) proceeds linearly, after a 5-min lag, for 30-40 min to produce as much as a 40% increase over the input DNA, (v) depends on RNA polymerase and gyrase as indicated by total inhibition by rifampicin and nalidixate, (vi) depends on replication proteins (e.g., dnaB protein and single-stranded DNA binding protein) as judged by specific antibody inhibitions, (vii) operates independently from protein synthesis, and (viii) depends on dnaA activity, as suggested by the inactivity of enzyme fraction from each of two dnaA temperature-sensitive mutant strains, and complementation (with a 15-fold overproduction of complementing activity) by a fraction from a strain containing the dnaA gene cloned in a multicopy plasmid . Resolution and analysis of factors that control the initiation of a chromosome cycle should become accessible through its enzyme system.

Proc Natl Acad Sci U S A, 1981 Dec, 78(12), 7350 - 4
A DNA fragment with an alpha-phosphorothioate nucleotide at one end is asymmetrically blocked from digestion by exonuclease III and can be replicated in vivo; Putney SD et al.; 2'-Deoxyadenosine 5'-O-(1-thiotriphosphate) (dATP{alpha S}) was introduced into the 3' ends of DNA restriction fragments with Escherichia coli DNA polymerase I to give phosphorothioate internucleotide linkages . Such "capped" 3' ends were found to be resistant to exonuclease III digestion . Moreover, the resistance to digestion is great enough that, under conditions used by us, just one strand of a double helix is digested by exonuclease III when a cap is placed at only one end; when digestion is carried to completion, this results in production of intact single strands . When digestion with exonuclease III is limited and is followed by S1 nuclease treatment, double-stranded DNA fragments asymmetrically shortened from just one side are produced . In this was thousands of nucleotides can be selectively removed from one end of a restriction fragment . In vitro introduction of phosphorothioate linkages into one end of a linearized replicative plasmid, followed by exonuclease III and S1 nuclease treatments, gives rise to truncated forms that, upon circularization by blunt-end ligation, transform E . coli and replicate in vivo.

Infect Immun, 1981 Dec, 34(3), 944 - 8
Evidence for a bladder cell glycolipid receptor for Escherichia coli and the effect of neuraminic acid and colominic acid on adherence; Davis CP et al.; The rat bladder epithelial cell receptors involved in mannose-sensitive adherence of Escherichia coli strains were studied . Sodium metaperiodate and lipase pretreatment of epithelial cells significantly reduced bacterial adherence to cells whereas trypsin and phospholipase C had a marginal or insignificant effect on adherence . Neuraminidase and colominic acid significantly increased adherence, whereas N-acetylneuraminic acid significantly reduced adherence . These data suggest that the rat bladder epithelial cell receptors involved in mannose-sensitive adherence are glycolipids . In addition, the data suggested that sialic acid on bladder epithelial cells acts as a nonspecific inhibitor of adherence, whereas colominic acid, a component of some E . coli K1 capsules, may act as a promoter of adherence.

Gene, 1981 Dec, 15(4), 395 - 403
Cloning a promoter that puts the expression of tetracycline resistance under the control of the regulatory elements of the mer operon; Bohlander FA et al.; We have sub-cloned from the Eco RI-H fragments of the IncFII plasmid R100 a 260-bp EcoRI fragment, using the promoter-cloning vehicle, pBRH4, (The Inc FII plasmid codes for the mer operon, and pBRH4 expresses tetracycline resistance only when the deleted tet promoter has been replaced by another sequence that can serve as a promotor) . With the 260-bp fragment inserted, the derivative plasmid, pFB4, directs the expression of tetracycline resistance only if there is a second plasmid in the strain that carries the merR-positive regulatory element . Under these conditions, the level of tetracycline resistance is directly proportional to the concentration of Hg2+ present in the medium . The 260-bp fragment also allows low-level constitutive expression of tet resistance when transactivated with merR mutants that have a "micro-constitutive" phenotype . The 260-bp mer promoter fragment contains a single HincII site; there is also but one HincII site in the EcoRI-H fragment of R100 from which the promoter fragment was derived . Restriction analysis of purified Eco RI-H DNA shows that the single HincII site is at 550 bp from the "right"terminus of the IS1b element, which is also present in the EcoRI-H fragment . Because of its biological activity and its location within the "H" fragment, this promoter is very likely a promoter for the structural genes of the operon.

Gene, 1981 Dec, 15(4), 331 - 42
Region- and strand-specific mutagensis of a recombinant plasmid; Giza PE et al.; Techniques were developed to mutagenize a single DNA strand in a specific region of the tetracycline-resistance (tetr) gene of the plasmid pKB280 that also carries the lambda repressor gene . Separate annealings of complementary single strands gave two isomeric, circular plasmids containing a 275-nucleotide, single-stranded region (gap) in the tetr gene . One of the isomeric, gapped plasmids was mutagenized specifically with sodium bisulfite such that an estimated 98% of the molecules had suffered at least one C to U conversion in the gap . The mutagenized gap was filled in with DNA polymerase . These molecules transformed Escherichia coli strain MM294 to lambda-immunity with the same frequency as unmutagenized, gap-filled pKB280 . Of the lambda-immune transformants, 32% were Tcr and 68% were Tcs . Restriction analysis of plasmids from some Tcs transformants showed losses of restriction sites within the gap and at the gap termini, but none outside the gap . No deletions were detected.

Gene, 1981 Dec, 15(4), 319 - 29
Versatile cloning vectors derived from the runaway-replication plasmid pKN402; Bittner M et al.; Two cloning vectors have been constructed employing runaway-replication mutants of plasmid R1 . One of these, pMOB45, carries tetracycline and chloramphenicol resistance . The other, pMOB48, carries chloramphenicol resistance, lacOP, and an assayable part of the lacPOZ operon . Both of these plasmids can be amplified to high levels by heat induction, which condition does not lead to inhibition of protein synthesis; thus the plasmid can produce large amounts of DNA and protein . In pMOB48, a unique BamHI site is present near the amino-terminus of the beta-galactosidase gene . Chimeras formed by the insertion of restriction fragments at this site can be detected on X-gal plates, and can be used for the lacIq-controlled expression of proteins which are fused to the amino-terminus of beta-galactosidase . Induction with IPTG at 40 degrees C leads to the synthesis of extremely high levels of proteins whose gene have been cloned into this site.

Gene, 1981 Dec, 15(4), 297 - 305
Construction of new vectors for cloning of promoters; Enger-Valk BE et al.; Vectors for cloning promoter-DNA fragments were derived from plasmid pBR313 (Bolivar et al., 1977) . These have several unique restriction sites and carry the trpA gene from Escherichia coli as a selective marker . The selection is based on an enhancement of the growth rate of those bacteria in which the expression of trpA is directed by the cloned promoter . The expression of trpA can be determined quantitatively, independently of the copy number of the vector, and should reflect the apparent strength of the promoter, since the DNA segment located before trpA contains translational stop signals in all three reading frames.

Cell, 1981 Dec, 27(2 Pt 1), 267 - 77
The cloning and reintroduction into animal cells of a functional CAD gene, a dominant amplifiable genetic marker; de Saint Vincent BR et al.; Rodent cells resistant to PALA, a specific inhibitor of the aspartate transcarbamylase activity of the multifunctional CAD protein, overproduce CAD as a result of amplification of the CAD gene . We cloned a functional CAD gene from Syrian hamster cells using a cosmid vector . Two independently isolated cosmids containing CAD genes have inserts 40 and 45 kb long . We introduced the cloned genes into CAD-deficient Chinese hamster ovary (CHO) cell mutants by fusing them with protoplasts of Escherichia coli containing the cosmids . We also introduced the cloned genes into wild-type CHO cells by selecting cells that became resistant to high concentrations of PALA following protoplast fusion . The transformants of the mutant and wild-type CHO cells contain multiple active copies of the donated Syrian hamster CAD genes . The cloned genes in three independent transformants are integrated into host-cell chromosomes at single locations identified by in situ hybridization . In two of these transformants, the genes are located in one X chromosome or in a chromosome resembling the X . In the third case, the genes are located in a small metacentric or rearranged chromosome.

Immunopharmacology, 1981 Dec, 3(4), 361 - 6
Restoration by cyclic guanosine monophosphate and extracellular calcium of butylated hydroxyanisole-suppressed primary murine thymus-dependent antibody response; Wess JA et al.; The effect of dibutyryl cyclic guanosine monophosphate (dbc-GMP) on butylated hydroxyanisole (BHA)-induced suppression of the primary in vitro thymus-dependent antibody response of BDF1 mouse spleen cultures was studied . When added at 0 hr relative to antigen addition, 1 mg of dbc-GMP (8 mM) restored by greater than 70% the BHA-inhibited primary immunoglobulin (Ig)M plaque-forming cell (PFC) response to sheep erythrocytes (SRBC) . The suppression was not reversed by the addition of 50 microgram of dibutryl cyclic adenosine monophosphate (dbc-AMP), which is known to reverse suppressor T-cell activity . The addition of 10 mM extracellular calcium (Ca2+) at the same time as antigen to BHA-inhibited cultures resulted in more than 80% restoration of the anti-SRBC PFC response . Quantitation of c-GMP by radioimmunoassay demonstrated that BHA lowered by 58% the c-GMP content of splenic lymphocytes and abrogated the ability of lipopolysaccharide of E . coli (LPS) to elevate c-GMP levels in splenic lymphocytes . The data suggest that BHA exerts its immunosuppressive effect on the primary in vitro antibody response by inhibiting guanylate cyclase activity and effectively lowering c-GMP levels; exogenous dbc-GMP and Ca2+ can freely reverse the immunosuppressive effect of BHA.

Antibiotiki, 1981 Dec, 26(12), 883 - 5
{Migration of the ampicillin transposon in enteropathogenic Escherichia}; Voskresenskii AM; Migration of Tn 1 from plasmid RP4 into the chromosome of enteropathogenic Escherichia (EPE) of serogroups O124 and O111 was studied . E . coli K 12 LC 411 (RP4) was used as the donor . It was shown that the transposition rate markedly differed depending on the period of ;the cell isolation from Tn 1 in the chromosome, i . e . during conjugation or after several subcultures of the transconjugants from the autonomic plasmid onto the selective media . During the conjugation process the migration rate of Tn 1 was equal to 0.5 and 0.8 per cell acquiring the plasmid . The transposition rate in the EPE carrying the R factor for a long period of time was 2.4 . 10(-2) and 1.4 . 10(-2) respectively for each serogroup . The above differences in the migration rate of Tn 1 were not concerned with changes in the environment.

J Virol, 1981 Dec, 40(3), 932 - 5
Molecular cloning of viral DNA from human genital warts; de Villiers EM et al.; The DNA of human papilloma virus type 6 (HPV 6) has been cloned in Escherichia coli K-12 by using pBR322 as vector . The DNA was cloned at the BamHI and EcoRI cleavage sites . This DNA was mapped by employing further restriction endonucleases and by terminal labeling . No major differences were noted as compared to HPV 6 DNA originating directly from a genital wart . The existence of at least two DNA subtypes (HPV 6a and 6b) became apparent.

J Gen Virol, 1981 Dec, 57(Pt 2), 285 - 96
Persistence and expression of Marek's disease virus DNA in tumour cells and peripheral nerves studied by in situ hybridization; Ross NL et al.; We have used cloned fragments of Marek's disease virus (MDV) DNA and in situ hybridization to search for virus DNA and study its expression in infected chick embryo fibroblasts (CEF), lymphoblastoid cell lines, tumours and neural lesions . DNA from the HPRS 16/att strain of MDV was cleaved with EcoRI endonuclease and several fragments were cloned in Escherichia coli using the vector PBR322 . Seven fragments ranging in size from 2.6 to 11 kbp representing approx . 25% of the MDV genome were labelled in vitro and annealed to EcoRI digests of DNA from infected cells and tumours following separation and transfer according to the Southern blotting procedure . Most of the selected MDV DNA fragments hybridized to fragments of corresponding sizes in EcoRI digests of DNA from cell lines and tumours and failed to hybridize to digests of uninfected chick cell DNA . In situ hydridization using 3H-labelled DNA with specific activity of 10(8) d/min/microgram as probe showed intranuclear MDV DNA in infected CEF, in every cell of two lymphoblastoid cell lines and in the majority of infiltrating or proliferating lymphoid cells found in type 'A' lesions of grossly enlarged peripheral nerves . Both intranuclear and cytoplasmic RNA were detected in cells that contained virus DNA . However, comparatively little virus RNA appears to be transcribed in cell lines and in infected tissues from the regions of virus DNA (25% of genome) used as probe in this study . Our results favour the hypothesis that the accumulation of lymphoid cells in nerves is not the result of an inflammatory response to infected nerve cells but is rather the consequence of proliferating transformed cells.

Biokhimiia, 1981 Dec, 46(12), 2250 - 6
{Mapping of the 16S rRNA regions in ribosomes capable of complementary binding of oligonucleotides}; Skripkin EA et al.; 16S rRNA regions have been mapped on the surface of the 30S ribosomal subunit of E . coli due to their ability to bind the statistical mixture of hexadeoxyribonucleotides and thus to be hydrolyzed with RNAase H . These regions were found to be located around 80, 996-998, 1044-1046, 1408-1410, 1423, 1484-1485, 1495, 1500-1506, 1531-1532 16S rRNA nucleotide residues.

J Natl Cancer Inst, 1981 Dec, 67(6), 1347 - 51
Age- and sex-related differences in antibody formation and blastogenic responsiveness of splenocytes from RIII mice developing virus-induced mammary adenocarcinoma; Specter S et al.; Inbred RIII mice, known to be infected neonatally with murine mammary tumor virus so that females develop mammary adenocarcinoma by 12-15 months of age, were examined with regard to antisheep red blood cell antibody responses at the cellular level . Female mice, 3-11 months old, compared to male mice of the same ages had consistent and significant depression of the antibody response of their splenocytes . Furthermore, female mice with adenocarcinoma showed an even greater depression of the antibody response . Spleen sizes were consistently increased in females as compared to those of male mice throughout the first year of life . The blastogenic responsiveness of the splenocytes to the B-cell mitogen Escherichia coli lipopolysaccharide and the T-cell mitogen phytohemagglutinin-P was not significantly different between male and female mice during the same periods, although the responses of the older tumor-bearing female mice to phytohemagglutinin-P were lower than those of non-tumor-bearing female mice . A complex relationship between age, sex, and immune responsiveness was evident in these mammary tumor virus-infected mice, which made it difficult to attribute a specific immune event to emergence of the mammary adenocarcinomas in the female as compared to male mice.

J Bacteriol, 1981 Dec, 148(3), 817 - 28
Plasmid-determined immunity of Escherichia coli K-12 to colicin Ia Is mediated by a plasmid-encoded membrane protein; Weaver CA et al.; The colicin Ia structural (cia) and immunity (iia) genes of plasmid pColIa-CA53 have been cloned into the cloning vector pBR322 . These two genes are closely linked, and both of them can be isolated on a deoxyribonucleic acid fragment approximately 4,800 base pairs long . An analysis of the polypeptides synthesized in ultraviolet-irradiated cells containing these cloned genes led to the conclusion that the iia gene product is a polypeptide with a molecular weight of approximately 14,500 . Insertion of transposon Tn5 into the iia gene led to a concomitant loss of the immune phenotype and the ability to produce this protein . Fractionation of ultraviolet-irradiated cells harboring a plasmid carrying the iia gene showed that the immunity protein is a component of the inner (cytoplasmic) membrane . Furthermore, the mechanism of immunity to colicin Ia appears to operate at the level of the cytoplasmic membrane . This conclusion is based on our finding that membrane vesicles prepared from colicin Ia-immune cells could be depolarized by colicins E1 and Ib but not by colicin Ia.

J Bacteriol, 1981 Dec, 148(3), 753 - 61
Isolation and characterization of an Escherichia coli mutant affected in the regulation of adenylate cyclase; Guidi-Rontani C et al.; A mutant, cyaR1, affecting regulation of adenylate cyclase expression or activity is described . It was obtained as a thermoresistant revertant of a strain harboring a thermosensitive transcription termination factor, rho (rho-15) . This mutant failed to synthesize adenosine 3',5'-phosphate and exhibited a carbohydrate-negative phenotype . A secondary mutation at the crp locus (crpC) restored the ability of the mutant to synthesize adenosine 3',5'-phosphate, enabled the expression of catabolite-sensitive operons, and conferred on the strain an extreme sensitivity to catabolite repression . In addition, we showed that the crpC mutation restored the pleiotropic carbohydrate-positive phenotype even in a delta cya background . We interpret this to mean that the adenosine 3',5'-phosphate receptor protein regulates negatively either the activity or synthesis of adenylate cyclase and that the cyaR1 mutation is either in a regulatory protein or a regulatory site of adenylate cyclase.

Gene, 1981 Dec, 16(1-3), 261 - 74
Antitermination and termination functions of the cloned nutL, N, and tL1 modules of coliphage lambda; Drahos D et al.; A plasmid containing a pp-galK operon was constructed to assay galK expression as a measure of transcriptional regulation by the cloned nutL, N and tL1 modules in a Rho+, Nus+ Escherichia coli host . Insertion of tL1 and rut, both carried on a lambda fragment located between the bamHI site and gene N, between the promoter and galK reduced expression of galK by 80--90% . Gene N alone, when controlled by pp, stimulated galK expression by about two-fold . Cloning of both gene N and nutL in the proper orientation resulted in about a 60% decrease in the tL1 termination . Additional tandem nutL sites increased efficiency of antitermination . A shortened nutL segment, containing only 25 bp of the original genomic nutL sequence, was found to have nearly equal ability to bring about antitermination . If the orientation of the nutL fragment is reversed, antitermination is abolished and the insert now displays a termination function . Termination efficiency is 60 to 73% for one to four tandem nutL modules in reverse orientation . Similarly, the inverted N module acts as a terminator, with 67% efficiency for one and 90% for two tandem inserts . Termination by inverted nutL or N fragments is probably unrelated to their normal functions, but indicates a fortuitous presence of a terminator sequence in the inverted orientation.

Gene, 1981 Dec, 16(1-3), 249 - 59
Isolation and fine structure organisation of an avian vitellogenin gene coding for the major estrogen-inducible mRNA; Wilks A et al.; Two phage lambda recombinant DNA clones covering the entire sequence of an avian vitellogenin gene, plus flanking regions, have been isolated from an erythrocyte DNA gene library and characterized by R-loop and restriction mapping . The total length of this avian vitellogenin gene is 23 kb . The cloned sequences flanking the gene at the 5' and 3' end are 7 and 3 kb, respectively . The total length of exons in the two clones is 6.7 kb (vitellogenin mRNA is 6.6 kb) . The gene is interrupted by at least 25 introns with a mean intron length of 940 bp . Some 6--10 additional very small introns may also be present but they were not observed reproducibly . The mean exon length is 250 bp . Restriction endonuclease digests of total liver genomic DNA and lambda recombinant DNA were also analyzed by electrophoresis . Southern blotting and hybridization with cloned vitellogenin cDNA . The results show an identity of organisation of this vitellogenin in the DNA from the two sources, thus ruling out a possible cloning artifact . In contrast to Xenopus vitellogenin we have found no evidence to suggest that avian vitellogenin is encoded by a small family of related genes.

Gene, 1981 Dec, 16(1-3), 141 - 8
The nucleotide sequence of the mouse embryonic beta-like y-globin messenger RNA as determined from cloned cDNA; Vanin EF et al.; We have determined the nucleotide sequence of two cloned cDNAs corresponding to the mRNA of mouse embryonic y2 globin . The combined overlapping sequences span a total of 480 bp, beginning at the codon corresponding to amino acido residue 21 and extending to the AATAAA sequence in the 3' untranslated region . Therefore, when the amino acid sequence encoded by the cDNA is combined with the available amino acid sequence, a complete y2 protein sequence can be obtained . Comparisons, at the nucleotide level, between the known beta- and beta-like globin sequences and the y2 sequence show that the embryonic, fetal-adult duplication occurred approx . 160 million years (MY) ago and that the embryonic-fetal duplication occurred approx . 100 MY ago.

Med Biol, 1981 Dec, 59(5-6), 368 - 73
Effect of polyamines on the fidelity of macromolecular synthesis; Abraham AK; Addition of polyamines to in vitro systems containing suboptimal concentrations of Mg2+ markedly stimulated protein and RNA synthesis . This stimulation is observed only within a marrow range of polyamine concentration . The extend of stimulation of RNA synthesis was dependent on assay conditions . Addition of spermidine to the wheat germ system not only stimulated poly(U) directed polyphenylalanine synthesis, but also reduced the misincorporation of leucine . MS2-coat protein synthesis, studied in an E.coli cell-free system using either one of the two glutamyl-tRNAs as the only source of glutamine, suggested that in the presence of spermidine, codon recognition by these two isoacceptor tRNA molecules was more stringent . From these results it is concluded that polyamines contribute to the specificity of codon/anticodon interactions and thereby increase the fidelity of protein synthesis.

Proc Natl Acad Sci U S A, 1981 Dec, 78(12), 7702 - 6
Antibody production by single, hapten-specific B lymphocytes: an antigen-driven cloning system free of filler or accessory cells; Vaux DL et al.; CBA mouse spleen cells were subjected to hapten affinity fractionation on thin layers of fluorescein (FLU)-gelatin . This procedure yields 97% B cells with varying FLU-binding avidities . One to 30 cells were placed in 10-microliter microcultures without any filler or accessory cells . the T-independent antigen polymerized flagellin coupled to FLU (FLU-POL) was ineffective in stimulating these cells to clonal proliferation or antibody production when used alone . Unpurified preparations rich in interleukins also failed to stimulate the cells . When specific antigen, but not in irrelevant hapten-POL, was combined with the interleukins, clonal proliferation was stimulated and most clones produced anti-FLU antibody-forming cells . The frequency of antibody-forming clones was only slightly lower than that in a system using antigen plus filler cells . In the absence of added interleukins, the mitogens Escherichia coli lipopolysaccharide plus dextran sulfate induced equivalent antibody production . However, a higher frequency of clonal proliferation was noted . Added interleukins did not aid these mitogen-driven responses . Such an antigen-dependent cloning system, free of filler and accessory cells, should permit more precise analysis of the respective roles of antigens and interleukins in the physiology of antibody-forming clone formation.

Eur J Biochem, 1981 Dec, 120(3), 487 - 95
A general secondary-structure model for procaryotic and eucaryotic RNAs from the small ribosomal subunits; Stiegler P et al.; A consensus on the folding of the Escherichia coli 16-S ribosomal RNA is emerging and several complete nucleotide sequences of small ribosomal subunit RNAs, covering diverse types of organisms and organelles, are now available . We therefore investigated the extent of both nucleotide sequence and secondary structure conservation that may exist between the E . coli 16-S RNA and other ribosomal RNAs . All the RNA molecules examined could be folded into secondary structure schemes that illustrated remarkable preservation of many structural motifs as well as striking nucleotide sequence conservation compared with the E . coli molecule . This study presents a unitary scheme for the structural organization of the small ribosomal subunit RNAs . The evolutionary constraints on both primary and secondary structures most likely reveal the basic role of some restricted RNA regions in the function of the ribosome.

Eur J Biochem, 1981 Dec, 121(1), 33 - 7
Cold-sensitive ribosome assembly in an Escherichia coli mutant lacking a single methyl group in ribosomal protein L3; Lhoest J et al.; Ribosomal protein methylation has been well documented but its function remains unclear . We have examined this phenomenon using an Escherichia coli mutant (prmB2), which fails to methylate glutamine residue number 150 of ribosomal protein L3 . This mutant exhibits a cold-sensitive phenotype: its growth rate at 22 degrees C is abnormally low in complete medium . In addition, strains with this mutation accumulate abnormal and unstable ribosomal particles; 50-S and 30-S subunits are formed, but at a lower rate . Once assembled, ribosomes with unmethylated L3 are fully active by several criteria . (a) Protein synthesis in vitro with purified 70-S prmB2 ribosomes is as active as wild-type using either a natural (R17) or an artificial {poly(U)} messenger . (b) The induction of beta-galactosidase in vivo exhibits normal kinetics and the enzyme has a normal rate of thermal denaturation . (c) These ribosomes are standard when exposed in vitro to a low magnesium concentration or increasing molarities of LiCl . Efficient methylation of L3 in vitro requires either unfolded ribosomes or a mixture of ribosomal protein and RNA . We suggest that the L3-specific methyltransferase may qualify as one of the postulated 'assembly factors' of the E . coli ribosome.

Eur J Biochem, 1981 Dec, 121(1), 233 - 5
Rotational diffusion of eosin-labeled pyruvate dehydrogenase complex of Escherichia coli; Visser AJ et al.; The enzymatically reduced lipoyl residues of the transacetylase component of the pyruvate dehydrogenase complex from Escherichia coli were labeled with eosin maleimide . Using eosin as triplet probe, triplet-triplet absorption dichroism measurements were performed to obtain rotational correlation times of the complex in the microsecond time domain . It was found that the hydrodynamic properties determined from the correlation times are in very good agreement with those obtained with other methods of different origin . The results can be fully explained by eosin molecules rotating with the whole complex, which consists of a mixture of heavy (60 S) and light (20 S) particles . Since no independent mobility could be detected it is suggested that the (charged) chromophoric group is folded against the protein surface . Labeling with excess eosin maleimide tends to destabilize the complex, since the longer correlation time (60 S) decreases and the contribution of the shorter correlation time (20 S) becomes more significant upon labeling.

J Bacteriol, 1981 Dec, 148(3), 941 - 9
Cloning and characterization of the gene for Escherichia coli tryptophanyl-transfer ribonucleic acid synthetase; Hall CV et al.; From a Clark-Carbon plasmid containing trpS, the structural gene for the tryptophanyl-transfer ribonucleic acid synthetase of Escherichia coli, we subcloned a 2.6-kilobase fragment that has trpS and its neighboring regions . The location and orientation of trpS in the deoxyribonucleic acid insert was determined by deoxyribonucleic acid sequencing . In vitro transcription experiments and S1 nuclease mapping studies with in vivo message established that transcription is initiated at the same site in vivo and in vitro, approximately 58 base pairs upstream from the trpS coding region . We also describe the construction of an inphase trpS-lacZ gene fusion that is under the control of the trpS promoter and encodes a hybrid protein with beta-galactosidase activity.

J Bacteriol, 1981 Dec, 148(3), 897 - 903
Tn9 and IS1 inserts in a ribosomal ribonucleic acid operon of Escherichia coli are incompletely polar; Brewster JM et al.; Transcription is known to be coupled to translation in many or all bacterial operons which code for proteins . In these operons, nonsense codons which prevent normal translation often result in premature termination of transcription (polarity) . However, efficient transcription of ribosomal ribonucleic acid operons (rrn operons) occurs, although rrn transcripts are not translated . It therefore seemed possible that insertion sequences and transposable elements which are polar in protein-coding operons might not be polar in rrn operons . Previously, it has been shown (E . A . Morgan, Cell 21:257-265, 1980) that Tn10 is incompletely polar in the rrnX operon . Here we show that the transposon Tn9 and the insertion sequence IS1 also incompletely polar in rrnX . In normal cells expression of sequences distal to the insertions can be detected by genetic methods . In ultraviolet-irradiated cells expression of distal sequences is about 80% of that observed in uninterrupted rrnX operons . These observations provide evidence that ribonucleic acid polymerase molecules beginning at rrnX promoters can read through Tn9 and IS1 and that, at least in ultraviolet-irradiated cells, read-through is very efficient.

J Bacteriol, 1981 Dec, 148(3), 782 - 7
Enhancement of ribosomal ribonucleic acid synthesis by deoxyribonucleic acid gyrase activity in Escherichia coli; Oostra BA et al.; The effect of the deoxyribonucleic acid (DNA) gyrase inhibitors coumermycin A1, novobiocin, and oxolinic acid on ribonucleic acid (RNA) synthesis in Escherichia coli was studied in vivo and in vitro . Preferential inhibition of ribosomal RNA (rRNA) synthesis was observed . No effect of oxolinic acid and coumermycin on rRNA synthesis was seen in mutants having a DNA gyrase which is resistant to these inhibitors . In a temperature-sensitive DNA gyrase mutant rRNA synthesis was decreased at nonpermissive temperatures . Thus, a functional DNA gyrase is required for rRNA synthesis . Purified DNA gyrase had no effect on rRNA synthesis in a purified system . However, DNA gyrase does show preferential stimulation of rRNA synthesis in a system supplemented with other proteins . Apparently, DNA gyrase stimulation of rRNA synthesis requires another protein.

Biochim Biophys Acta, 1981 Nov 27, 656(1), 123 - 7
Efficiency of T4 DNA ligase-catalyzed end joining after S1 endonuclease treatment on duplex DNA containing single-stranded portions; Shishido K et al.; Covalently closed-circular, superhelical SV4O DNA was used in all experiments . EcoRI endonuclease- and HpaII endonuclease-generated unit-length linear duplex DNAs were digested with S1 endonuclease under the conditions where single-stranded CNA was completely converted into the acid-soluble form . These were subjected to an end-to-end joining test with T4 DNA ligase . The ligation efficiency was significantly lower than that of the flush-ended linear duplex DNAs which were generated by both HpaI endonuclease digestion and the matching up of EcoRI-generated sticky end with Escherichia coli DNA polymerase I (Klenow fraction) . However, the ligation efficiency of the S1-treated DNAs increased up to same level as the flush-ended DNA upon treatment with E . coli DNA polymerase I . Similar results were obtained in the case of S1 -generated unit-length linear duplex DNA . S1 does cleave both strands of superhelical DNA at unbasepaired sites.

J Biol Chem, 1981 Nov 25, 256(22), 11873 - 9
Chloroplast ribosome structure . Electron microscopy of ribosomal subunits and localization of N6,N6-dimethyladenosine by immunoelectronmicroscopy; Trempe MR et al.; Ribosomal subunits from the chloroplasts of Alaskan peas have been studied by immunoelectronmicroscopy . Electron micrographs of negatively stained small and large ribosomal subunits show particles of similar size and in the same characteristic projections described for the ribosomal subunits of Escherichia coli (Lake, J . A . (1976) J . Mol . Biol . 105, 131-159), although minor structural differences are apparent . High pressure liquid chromatographic analysis shows the modified nucleoside N6,N6-dimethyladenosine is conserved in chloroplast 16 S ribosomal RNA, presumably as two successive residues near the 3' end . Antibodies directed against N6,N6-dimethyladenosine were allowed to react with chloroplast 30S ribosomal subunits . Electron microscopy showed individual subunit-antibody complexes and pairs of ribosomal subunits cross-linked by a single antibody . In 94% of the complexes observed, antibody contact was consistent with a dimethyladenosine localization near the end of the small subunit platform, in an area of subunit contact in the 70 S ribosome . This localization is analogous to the placement of N6,N6-dimethyladenosine in the E . coli ribosome (Politz, S . M., and Glitz, D . G . (1977) Proc . Natl . Acad . Sci . U . S . A . 74, 1468-1472).

J Biol Chem, 1981 Nov 25, 256(22), 11804 - 8
Purification and reconstitution of functional lactose carrier from Escherichia coli; Newman MJ et al.; The lactose carrier protein of Escherichia coli was purified by a simple procedure employing differential solubilization and ion-exchange chromatography and reconstituted into liposomes by octylglucoside dilution . The proteoliposomes exhibited both membrane potential-driven lactose transport and lactose counterflow . Furthermore, the purified protein was identified as the product of the lac y gene . These and other results demonstrate that the lactose carrier is the only polypeptide species essential for energy-coupled lactose transport and counterflow.

J Biol Chem, 1981 Nov 25, 256(22), 11595 - 9
Inducer and anti-inducer interactions with the lac repressor seen by nuclear magnetic resonance changes at tyrosines and tryptophans; Boschelli F et al.; The effects of binding inducer and anti-inducer of the Escherichia coli lac operon to the lac repressor were examined by taking advantage of fluorine-19 NMR . The fluorine nucleus was biosynthetically incorporated into the lac repressor with either 5-fluorotryptophan or 3-fluorotyrosine . It is suggested that these small effector molecules influence the operator-binding properties of the tetrameric lac repressor by altering the intersubunit relationships in the protein.

J Biol Chem, 1981 Nov 25, 256(22), 11428 - 33
Kinetics of aspartate transcarbamylase from Escherichia coli for the reverse direction of reaction; Foote J et al.; The reverse reaction of aspartate transcarbamylase in which phosphate or arsenate is first coupled to carbamyl aspartate, followed by elimination of aspartate, has been studied under conditions in which one product, aspartate, is removed . Aspartate is converted to oxalacetate by glutamate-oxalacetate transaminase, and the resulting oxalacetate is converted to malate by the NADH, NAD+ oxidoreductase enzyme malate dehydrogenase . Phosphate and carbamyl aspartate saturation curves are nonsigmoidal . The transition state analogue, N-phosphonacetyl-L-aspartate, activates this reverse reaction substantially . Reverse kinetic parameters of the Haldane type are characteristic of the T-state and correlated with the parameters of the usual forward reaction of the T-state . Phosphate and carbamyl aspartate do not alter the thiol reactivity or sedimentation coefficient of the enzyme . These five results indicate that, under the conditions of these experiments, the reverse reaction does not cause the allosteric transition . In a new assay for the forward reaction we couple phosphate production with NADP reduction using phosphorylase a, phosphoglucomutase, and glucose-6-phosphate dehydrogenase.

Nucleic Acids Res, 1981 Nov 25, 9(22), 6083 - 92
Selective inhibition by harmane of the apurinic apyrimidinic endonuclease activity of phage T4-induced UV endonuclease; Warner HR et al.; 1-Methyl-9H-pyrido-{3,4-b}indole (harmane) inhibits the apurinic/apyrimidinic (AP) endonuclease activity of the UV endonuclease induced by phage T4, whereas it stimulates the pyrimidine dimer-DNA glycosylase activity of that enzyme . E . coli endonuclease IV, E . coli endonuclease VI (the AP endonuclease activity associated with E . coli exonuclease III), and E . coli uracil-DNA glycosylase were not inhibited by harmane . Human fibroblast AP endonucleases I and II also were only slightly inhibited . Therefore, harmane is neither a general inhibitor of AP endonucleases, nor a general inhibitor of Class I AP endonucleases which incise DNA on the 3'-side of AP sites . However, E . coli endonuclease III and its associated dihydroxythymine-DNA glycosylase activity were both inhibited by harmane . This observation suggests that harmane may inhibit only AP endonucleases which have associated glycosylase activities.

Nucleic Acids Res, 1981 Nov 25, 9(22), 6069 - 82
The primary structure of bovine satellite 1.715; Gaillard C et al.; The primary structure of the 1402 bp repeat unit of bovine satellite 1.715 has been determined using a dimer inserted at the SalI site of plasmid pBR322 and cloned in E . coli . In contrast with bovine satellites 1.706, 1.720b and 1.711a, the 1.715 satellite has a complex sequence with no obvious internal short prototype repeat . The sequence consists however of repeats ranging in length from 6 to 13 nucleotides . In addition, the hexanucleotide, AGATGA, present in the prototype sequences of satellites 1.706, 1.720b and 1.711a, is found in satellite 1.715 in repeats as long as, or longer than, 8 nucleotides, establishing a homology link among those satellites on one hand and satellite 1.715 (and the related satellite 1.711b) on the other . In turn, this suggests a common evolutionary origin . A comparison of the maps for 15 restriction enzymes of cloned and uncloned satellite indicates very little sequence divergence among the repeat units of the latter, most of the differences being due to methylation.

Nucleic Acids Res, 1981 Nov 25, 9(22), 6031 - 46
The interaction of RNA polymerase II with non-promoter DNA sites; Chandler DW et al.; Various complexes formed between purified RNA polymerase II and simian virus 40 DNA have been characterized with respect to rates of formation, rates of dissociation, and initial velocity of RNA synthesis . Two different types of complexes can form on intact DNA templates . One of these is formed rapidly, but is quite labile; the other forms more slowly, but is moderately stable once formed . The introduction of a single strand break into DNA leads to rapid and stable complex formation, and thus is expected to create the favored binding site . The observed properties of these complexes provide a general framework for describing the interactions of RNA polymerase II at non-promoter DNA sites . This framework appears to be similar to that established for Escherichia coli RNA polymerase interactions, suggesting that the fundamental mode of non-promoter DNA binding is similar for the bacterial, plant, and mammalian enzymes.

J Biol Chem, 1981 Nov 25, 256(22), 11911 - 6
Mosaic structure and mRNA precursors of uteroglobin, a hormone-regulated mammalian gene; Snead R et al.; The synthesis of uteroglobin in the rabbit uterus is induced by progesterone and is repressed by estrogen which has an over-riding effect over the inducer . The dual hormonal control system offers an excellent model for studying hormonal regulation of mammalian gene expression . Using a full-length uteroglobin cDNA clone as a specific hybridization probe, recombinant lambda phages containing the entire chromosomal uteroglobin gene have been isolated from a rabbit genomic DNA library . Electronmicroscopic analysis of hybrid molecules formed between the chromosomal uteroglobin gene and uteroglobin mRNA indicated the presence of 2 intervening sequences within this gene . The mosaic structure of the uteroglobin gene has been substantiated by detailed restriction mapping and Southern hybridization . The gene is 3.0 kilobases in length to code for a mature mRNA of 465 nucleotides . Northern hybridization of poly(A)-containing RNA from 4-day-pregnant rabbit uterus with the full-length cDNA clone revealed the presence of uteroglobin mRNA precursors . The size of the largest precursor RNA species detected by the cDNA clone is the same as the entire chromosomal uteroglobin gene . The fidelity of the precursor RNAs was established by their ability to hybridize with specific intervening sequence probes . Thus the entire uteroglobin gene is expressed into primary RNA transcripts, which are subsequently processed into mature mRNA molecules by splicing.

J Biol Chem, 1981 Nov 25, 256(22), 11905 - 10
Evidence for two functional gal promoters in intact Escherichia coli cells; Aiba H et al.; We have used an S1 mapping assay to demonstrate that the mRNA transcripts of the Escherichia coli galactose operon found in intact E . coli cells with a defect in adenylate cyclase or the cyclic AMP receptor protein contain at their 5' end about five nucleotides more than the gal mRNA molecules made in wild type cells . The same difference between gal RNA synthesized in vitro in the absence of cyclic AMP or cyclic AMP receptor protein and gal RNA made in the presence of these factors is detected by this assay . Our results strongly suggest that the same two overlapping promoters, which we previously identified by in vitro transcription of gal DNA fragments, also control the expression of the galactose operon in intact cells . The intracellular levels of cyclic AMP determine which promoter is utilized.

Nucleic Acids Res, 1981 Nov 25, 9(22), 6153 - 66
Antiviral activities of hybrids of two major human leukocyte interferons; Weck PK et al.; Four hybrid human leukocyte interferon (LeIF or IFN-alpha) genes have been constructed by in vitro recombination of LeIF-A (IFN-alpha 2) and LeIF-D (IFN-alpha 1) genes at common restriction endonuclease sites located within their coding regions . These hybrid genes have been expressed in E . coli under trp promoter control . The interferons produced {LeIF-AD (BglII), -AD (PvuII), -DA (BglII), -DA (PvuII)} have antiviral properties distinct from the parental molecules LeIF-A and -D, varying considerably in their abilities to inhibit plaque formation by different viruses in a range of mammalian cells . All six of the cloned LeIFs exhibit the heat stability, pH 2 stability and antigenic specificity of natural leukocyte interferons.

J Biol Chem, 1981 Nov 25, 256(22), 11651 - 6
Correlation between RNA synthesis and basal level guanosine 5'-diphosphate 3'-diphosphate in relaxed mutants of Escherichia coli; Lagosky PA et al.; Through the use of a new nucleotide extraction procedure, we had previously shown that relaxed mutants of Escherichia coli exhibit a unique response to amino acid starvation (Lagosky, P . A., and Chang, F . N . (1980) J . Bacteriol . 144, 499-508) . The basal level amounts of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) in both relA and phenotypically relaxed relA+ rplK (relC) strains were shown to decrease at the onset of amino acid limitation and to remain severely depressed throughout the course of the starvation . Upon resupplementation of amino acid-starved relaxed mutants, the production of ppGpp resumes and results in the temporary overaccumulation of this nucleotide beyond its original basal level amount . We now show that the basal level ppGpp content of relaxed bacteria, as well as its subsequent fluctuations in response to amino acid starvation, is inversely correlated with the initial rates of RNA synthesis in these strains . The ability of ppGpp to control the rate of protein synthesis in relA mutants was also examined . It was observed that ppGpp had no apparent direct effect on the initial rates of protein synthesis in relA mutants . The constant inverse correlation which exists between ppGpp content in relA mutants, and their rates of RNa synthesis provide evidence which indicates that basal level ppGpp synthesis has definite physiological significance . It also suggests that the synthesis of basal level ppGpp might be an absolute requirement needed for normal bacterial growth.

J Biol Chem, 1981 Nov 25, 256(22), 11569 - 73
Selective inhibition of RNase H by dextran; Dirksen ML et al.; Ordinarily, ribonuclease H hydrolyzes poly(rA) . poly(dT) and phiX174DNA-RNA at equal rates . Here we show that in the presence of dextran, the degradation of poly(rA) . poly(dT) is inhibited, while that of phi 174DNA-RNA is not . A similar inhibition by sucrose is found to be due to trace contamination of dextran in the sucrose . Ribose, deoxyribose, and a number of other saccharides fail to inhibit RNase H . In experiments where the two substrates are presented in the presence of the inhibitor, the kinetics indicates that both molecules are recognized by the enzyme, but only the phi X174DNA-RNA is degraded . That is, dextran does not interfere with the recognition site, but rather blocks hydrolysis . It is proposed that the ability of dextran to confer selectivity toward different substrates reveals a potential regulatory mechanism for RNase H activity which may represent a control step in the initiation of DNA synthesis.

Biochemistry, 1981 Nov 24, 20(24), 6929 - 48
Diffusion-driven mechanisms of protein translocation on nucleic acids . 1 . Models and theory; Berg OG et al.; Genome regulatory proteins (e.g., repressors or polymerases) that function by binding to specific chromosomal target base pair sequences (e.g., operators or promoters) can appear to arrive at their targets at faster than diffusion-controlled rates . These proteins also exhibit appreciable affinity for nonspecific DNA, and thus this apparently facilitated binding rate must be interpreted in terms of a two-step binding mechanism . The first step involves free diffusion to any nonspecific binding site on the DNA, and the second step comprises a series of protein translocation events that are also driven by thermal fluctuations . Because of nonspecific binding, the search process in the second step is of reduced dimensionality (or volume); this results in an accelerated apparent rate of target location . In this paper we define four types of processes that may be involved in these protein translocation events between DNA sites . These are (i) "macroscopic" dissociation--reassociation processes within the domain of the DNA molecule, (ii) "microscopic" dissociation--reassociation events between closely spaced sites in the DNA molecule, (iii) "intersegment transfer" (via "ring-closure") processes between different segments of the DNA molecule, and (iv) "sliding" along the DNA molecule . We present mathematical and physical descriptions of each of these processes, and the consequences of each for the overall rate of target location are worked out as a function of both the nonspecific binding affinity between protein and DNA and the length of the DNA molecule containing the target sequence . The theory is developed in terms of the Escherichia coli lac repressor--operator interaction since data for testing these approaches are available for this system {Barkley, M . (1981) Biochemistry 20, 3833; Winter, R . B., & von Hippel, P . H . (1981) Biochemistry (second paper of three in this issue); Winter, R . B., Berg, O . G., & von Hippel, P . H . (1981) Biochemistry (third paper of three in this issue)} . However, we emphasize that this approach is general for the analysis of mechanisms of biological target location involving facilitated transfer processes via nonspecific binding to the general system of which the target forms a small part.

Biochemistry, 1981 Nov 24, 20(24), 6961 - 77
Diffusion-driven mechanisms of protein translocation on nucleic acids . 3 . The Escherichia coli lac repressor--operator interaction: kinetic measurements and conclusions; Winter RB et al.; The association and dissociation kinetics of the Escherichia coli lac repressor--operator (RO) complex have been examined as a function of monovalent ion concentration and operator-containing DNA fragment length in order to investigate the mechanisms used by repressor in locating (and dissociating from) the operator site . Association rate constants (ka) measured with an 80- or a 203-base-pair lac operator containing DNA fragment are 3--5-fold smaller than those determined with a 6700-base-pair operator fragment or with intact lambda plac5 DNA (50000 base pairs) at all salt concentrations tested . At salt concentrations less than approximately 0.1 M KCl, association rate constants to all operator-containing DNA fragments (except lambda plac5 DNA) are insensitive to variations in salt concentration, but the limiting low salt value of ka appears to depend upon operator-containing DNA length . The value of ka for lambda plac5 DNA decreases significantly from the approximately 0.1 M KCl maximum at low salt . Above approximately 0.1 M KCl, repressor--operator association rate constants for all operator-containing DNA substrates tested show a similar decrease with increasing salt concentration, which does not appear to depend upon the length of the DNA molecule (except for the very small DNA fragments) . In contrast to the association reaction, kd, the dissociation rate constant, decreases linearly (on a log kd vs . log {KCl} plot) with decreasing salt concentration over virtually the entire salt concentration range studied (0.05--0.2 M KCl) . These results are consistent with the explanation of the unusually fast association kinetics for this system in terms of a two-step model in which repressor initially diffuses to a nonoperator DNA binding site (forming an RD complex) and then rapidly "scans" (in a locally correlated fashion) adjacent sites until the operator is located or the repressor dissociates from the chain . Dissociation of the RO complex follows the same two-step process in reverse . Quantitative comparisons are made between these results and the theoretical predictions of the two facilitating translocation mechanisms (one-dimensional "sliding" along the DNA double helix and direct transfer between DNA segments) developed in the first paper of this series {Berg, O . G., Winter, R . B., & von Hippel, P . H . (1981) Biochemistry (first paper of three in this issue)} . We conclude that the experimental data for the "faster-than-diffusion-controlled" interaction of repressor and operator can be quantitatively modeled by a two-step process in which sliding is the dominant transfer mechanism . Molecular models of the initial nonspecific binding event (including "hopping") as well as sliding and interchain transfer are discussed, and the possible roles of facilitated translocation mechanisms of the diffusion-driven type in this and other in vitro and in vivo protein--nucleic acid interaction processes are considered.

Vet Rec, 1981 Nov 21, 109(21), 461 - 3
Inheritance of Escherichia coli K88 adhesion in pigs: identification of nonadhesive phenotypes in a commercial herd; Snodgrass DR et al.; A method for preparing fragments of brush border from the small intestinal epithelial cells of pigs was modified so that specimens as small as 3 mm X 3 mm could be used . This modification also allowed more rapid preparation and the brush borders thus prepared adhered specifically to K88+ but not K88- Escherichia coli . At slaughter 12.2 per cent of 459 bacon weight pigs from a commercial herd were of nonadherent phenotype . Litters containing only nonadherent pigs were identified . Parents of these litters and siblings intended for breeding stock replacements could be identified as probably also being of nonadherent phenotype and this was confirmed by examining biopsy samples obtained by enterotomy from the siblings.

Science, 1981 Nov 20, 214(4523), 916 - 9
Cloned poliovirus complementary DNA is infectious in mammalian cells; Racaniello VR et al.; A complete, cloned complementary DNA copy of the RNA genome of poliovirus was constructed in the Pst I site of the bacterial plasmid pBR322 . Cultured mammalian cells transfected with this hybrid plasmid produced infectious poliovirus . Cells transfected with a plasmid which lacked the first 115 bases of the poliovirus genome did not produce virus.

Nature, 1981 Nov 19, 294(5838), 217 - 23
lambda Repressor and cro--components of an efficient molecular switch; Johnson AD et al.; In a lysogen, most genes of phage lambda are repressed; in response to a transient induction signal, they are efficiently switched on . The switch, which consists in part of a tripartite operator to which two regulatory proteins bind, depends not only on DNA--protein interactions, but also on effects transmitted from one DNA-bound protein to another . lambda Exemplifies a strategy that facilitates efficient switching between two physiological states in response to a transient signal.

Nucleic Acids Res, 1981 Nov 11, 9(21), 5845 - 54
A minimal mechanism for abortive initiation of transcription of T7 DNA; Smagowicz W et al.; A steady state kinetic analysis of product inhibition in abortive initiation of transcription at promoters A1 and A3 of T7 DNA was carried out . The obtained kinetic data are in agreement with a mechanism of ordered product release with pppApU being released first from the central complex . A minimal general mechanism for abortive initiation satisfying the kinetic results is given . The stimulation of transcription of T7 DNA by pppApU at low substrate concentrations was investigated.

Nucleic Acids Res, 1981 Nov 11, 9(21), 5671 - 8
The nucleotide sequence of the promoter and the amino-terminal region of alkaline phosphatase structural gene (phoA) of Escherichia coli; Kikuchi Y et al.; The promoter and the amino-terminal region of phoA, the structural gene for alkaline phosphatase of Escherichia coli K12, was cloned by using a promoter cloning vector pMC1403 . The nucleotide sequence of the cloned fragment has been determined . A sequence encoding the amino-terminal portion of mature alkaline phosphatase is found and it is preceded by a sequence encoding the signal peptide . The signal peptide consists of 21 amino acids; Met-Lys-Gln-Ser-Thr-Ile-Ala-Leu-Ala-Leu-Leu-Pro-Leu-Leu-Phe-Thr-Pro-Val-Thr-Lys-Ala . The translation initiation codon is GUG, which is preceded by the Shine-Dalgarno sequence GGAG . Upstream to these sequences, there is a typical procaryotic promoter . TATAGTC for the Pribnow box . Around the Pribnow box, there are several dyad symmetrical sequences, which may probably be concerned with the regulation of this gene.

Nucleic Acids Res, 1981 Nov 11, 9(21), 5623 - 43
The structure and function of the regulatory elements of the Escherichia coli uvrB gene; van den Berg E et al.; The construction and properties of recombinant plasmids carrying the Escherichia coli uvrB gene, including its transcriptional- and translational regulatory elements, is reported . The DNA sequence of the region, which governs the expression of the uvrB gene, has been determined . Within this sequence two non-overlapping DNA segments match the model sequence for Escherichia coli promoters (1) . The '-10 regions' and the '-35 regions' of the proposed uvrB promoters are, respectively, 5'TAAAAT (P1), 5'TATAAT (P2) and 5'TTGGCA (P1), 5'GTGATG (P2) . The existence and the position of these promoters has been established by elimination of one promoter (P2), using molecular cloning procedures, by length measurements of in vitro synthesized 'run-off' transcripts and by protection of the uvrB regulatory region for S1 nuclease digestion using in vivo made RNA . Potential sites of interaction within the uvrB regulatory region with regulatory proteins, such as the LexA protein (2) and the UvrC protein (3) are discussed.

Nucleic Acids Res, 1981 Nov 11, 9(21), 5507 - 20
Nucleotide sequence of the ribosomal RNA gene of Physarum polycephalum: intron 2 and its flanking regions of the 26S rRNA gene; Nomiyama H et al.; The 26S ribosomal RNA gene of Physarum polycephalum is interrupted by two introns, and we have previously determined the sequence of one of them (intron 1) (Nomiyama et al . Proc.Natl.Acad.Sci.USA 78, 1376-1380, 1981) . In this study we sequenced the second intron (intron 2) of about 0.5 kb length and its flanking regions, and found that one nucleotide at each junction is identical in intron 1 and intron 2, though the junction regions share no other sequence homology . Comparison of the flanking exon sequences to E . coli 23S rRNA sequences shows that conserved sequences are interspersed with tracts having little homology . In particular, the region encompassing the intron 2 interruption site is highly conserved . The E . coli ribosomal protein L1 binding region is also conserved.

Biochemistry, 1981 Nov 10, 20(23), 6584 - 8
Transcription of the major Drosophila heat-shock genes in vitro; Craine BL et al.; Active eukaryotic genes are more accessible to some proteins than bind DNA than are inactive genes . In order to probe the accessibility of the Drosophila heat-shock genes we have isolated nuclei from Drosophila tissue culture cells and have used these nuclei as templates for Escherichia coli RNA polymerase . With nuclei isolated from cells that had not been heat shocked, the synthesis of heat-shock RNA was not detected by hybridization to a DNA clone containing sequences from the major heat-shock region . In contrast, approximately 0.22% of the RNA synthesized in nuclei isolated from cells that had been previously heat shocked hybridized to the heat-shock clone . The synthesis of heat-shock RNA was DNA dependent, was sensitive to rifampicin and to actinomycin D, and represented a 70-fold enrichment over random transcription of the Drosophila genome . Transcription showed an extraordinary preference for a region 5' distal to the structural gene . These results demonstrate that preferential transcription by the bacterial RNA polymerase is indicative of the active state of Drosophila genes.

J Biol Chem, 1981 Nov 10, 256(21), 10774 - 7
EPR studies of the Mn(II) complex with elongation factor Tu and GDP Identification of oxygen ligands to Mn(II) by observation of 17O superhyperfine coupling; Eccleston JF et al.; The coordination sphere of Mn(II) in the complex with GDP and elongation factor Tu from Escherichia coli has been probed by EPR spectroscopy with 17O-labeled ligands . Inhomogeneous broadening in the EPR signals for Mn(II) due to unresolved superhyperfine coupling to the 17O nucleus was used to identify directly bound oxygen ligands . Results with GDP selectively enriched with 17O either in the alpha-phosphate or in the beta-phosphate revealed that GDP was a beta-monodentate ligand for Mn(II) in the complex with the protein . Results with 17O-enriched water showed that two water molecules are coordinated to the Mn(II) . The EPR spectrum for the complex is characteristic of octahedral coordination for Mn(II) . Hence, three ligands from the protein are required to complete the sextet of ligands.

Biochim Biophys Acta, 1981 Nov 5, 677(3-4), 358 - 62
Effect of unusual guanosine nucleotides on the activities of some Escherichia coli cellular enzymes; Pao CC et al.; Unusual guanosine nucleotides guanosine 5'-diphosphate 3'-diphosphate (ppGpp, also known as MSI) and guanosine 5'-diphosphate 3'-monophosphate (ppGp, also known as MSIII) accumulate to high concentrations in wild-type cells of Escherichia coli during amino acid starvation . We reported here that both nucleotides strongly inhibit the activity of enzymes IMP dehydrogenase and adenylosuccinate synthetase, the first enzymes of the guanylate and adenylate biosynthetic pathways . In both cases, ppGP (MSII) is a stronger inhibitor than ppGpp (MSI) . On the other hand, these two nucleotides exhibited opposite effects on the activity of phosphoenolpyruvate carboxylase, the enzyme that utilizes phosphoenolpyruvate . At their respective physiological concentrations, the activity of phosphoenolpyruvate carboxylase is activated by ppGpp and inhibited by ppGp.

Carbohydr Res, 1981 Nov 2, 97(1), 105 - 12
Structural studies of the O-specific side-chain of the lipopolysaccharide from Escherichia coli O 55; Lindberg B et al.; The structure of the O-specific side-chains of the lipopolysaccharide from Escherichia coli O 55 has been investigated, methylation analysis, specific degradations, and n.m.r . spectroscopy being the principal methods used . It is concluded that the O-specific side-chains are composed of pentasaccharide repeating-units having the following structure {where Col stands for colitose (3,6-dideoxy-L-xylo-hexose)}.(See formula in text).

J Dairy Sci, 1981 Nov, 64(11), 2258 - 61
Concentrations of glucocorticoids, bovine serum albumin, and somatic cells in mastitic milk; Fox LK et al.; Milk glucocorticoids, bovine serum albumin, and somatic cells were measured during experimental mastitis . Six cows were allotted evenly into injected and control groups . Injected cows received a single intramammary infusion of .5 mg sterile Escherichia coli endotoxin in a quarter chosen at random . Control cows were untreated . Milk quarter samples were taken from all cows at both milkings and 9 h following midnight milking 1 day prior to glucocorticoid concentrations occurred 9 h postinjection and were 17.5 ng/ml (+/- 10.2), and 4.4 ng/ml (+/- 2.3) in injected and noninjected quarters of the same cows . In injected cows glucocorticoid concentrations returned to baseline within 36 h . Glucocorticoids in control quarters were .4 ng/ml (+/- .3) . Peak bovine serum albumin was 27.8 ng/ml (+/- 22.8) and .56 bg/ml (+/- .09) in injected and noninjected quarters in the same cows as compared to a baseline of .2 mg/ml (+/- .04) in control cows . In injected quarters, bovine serum albumin remained elevated for 4 days, and somatic cells were elevated for 6 days after injection . Glucocorticoids, bovine serum albumin, and somatic cells were elevated markedly in injected quarters, and unlike concentrations of bovine serum albumin and somatic cells, elevated glucocorticoids in injected quarters were short lived.

J Inorg Biochem, 1981 Nov, 15(3), 223 - 31
Alterations in ribonucleic acid synthesis by chromium (III); Okada S et al.; The effect of Cr(III) on in vitro RNA synthesis directed by DNA and chromatin isolated from mouse liver was investigated in comparison with other inorganic metals . At 1 mM, CrCl3 significantly stimulated RNA synthesis when incubated with DNA or chromatin prior to the addition of Escherichia coli RNA polymerase, while other metals inhibited it . This stimulation by Cr(III) was caused even at 1 muM CrCl3 on either DNA- or chromatin- directed RNA synthesis . The Cr(III)-complexes of DNA and chromatin also showed significantly enhanced template activities . On the other hand, when RNA polymerase was preincubated with Cr(III), a remarkable inhibition was observed in RNA synthesis directed by both DNA and chromatin . In isolated mouse liver nuclei with endogenous RNA polymerases, Cr(III) stimulated Mg2+-activated RNA synthesis but not Mn2r-(NH4)2SO4-activated synthesis . These results suggest that Cr(III) may alter or regulate gene expressions in mammals.

Gene, 1981 Nov, 15(2-3), 285 - 8
Cloning of mouse beta-casein gene sequences; Mehta NM et al.; Casein messenger RNAs (mRNAcsn) were purified from lactating mammary glands of BALB/c mice and used as a starting material for cloning of casein gene sequences . Double-stranded casein cDNA (ds-cDNAcsn) was prepared and blunt-end ligated to HindIII-specific DNA linker molecules . After digestion with HindIII, the dsDNAcsn was inserted into the HindIII site of plasmid pBR322, using T4 DNA ligase . Escherichia coli strain RH202 was transformed with the hybrid plasmids, and transformants were selected for resistance to ampicillin . Electrophoresis of HindIII-digested hybrid plasmid DNAs, followed by Southern transfer and hybridization to {32P}cDNAcsn, revealed that one of the hybrid-plasmid-containing colonies, designated pCas51, contained a 400-bp insert which hybridized to the {32P}cDNAcsn . Purification of the individual casein mRNAs (mRNAcsn alpha, beta, and gamma) and solution hybridization of nick-translated insert DNA to each of these revealed that pCas51 contained sequences complementary primarily to mRNAcsn beta.

J Gen Microbiol, 1981 Nov, 127, pt 1, 19 - 25
A role for threonine deaminase in the regulation of alpha-acetolactate biosynthesis in Escherichia coli K12; Squires CH et al.; The flow of carbon to alpha-acetolactate is Escherichia coli K12 is shown to involve the endogenous pool of alpha-ketobutyrate (alpha-KB) . In vivo, the acetohydroxy acid synthase (AHAS) isoenzymes have an affinity for alpha-KB sufficiently high that alpha-acetolactate production is severely limited when alpha K-B is supplied exogenously . The ability of threonine deaminase to make alpha-KB is correlated with the synthesis of the AHAS isoenzymes . Mutations in ilvA that alter the catalytic and allosteric properties of threonine deaminase affect alpha-KB production and the expression of the AHAS isoenzymes in a direct way . The ilv A538 mutation results in a feedback-hypersensitive threonine deaminase ans slow alpha-KB and AHAS production . A spontaneous revertant of an ilvA538 strain expressing a feedback-resistant threonine deaminase produces alpha-KB and AHAS more quickly . A physiological role for the activator (valine) site on threonine deaminase is proposed and valine is shown to increase alpha-KB production in vivo . Valine can thus regulate its own biosynthetic pathway without jeopardizing the production of isoleucine . The physiological implications of the role of alpha-KB in the biosynthesis of acetolactate are discussed.

J Biochem (Tokyo), 1981 Nov, 90(5), 1437 - 44
Stringent control of intermediary metabolism in Escherichia coli: pyruvate excretion by cells grown on succinate; Kodaki T et al.; A large amount of pyruvate was excreted into the medium by CP78 (rel+) cells grown on succinate when they were starved for amino acids . In contrast, no such excretion was observed with CP79 (rel-) cells . This phenomenon was also seen with two other isogenic pairs of strains: NF161 (rel+) and NF162 (rel-), and 10B601 (rel+) and 10B602 (rel-) . Besides succinate, L-malate, and fumarate were effective carbon sources for the excretion, but glucose, glycerol, and acetate were not . When DL-lactate was used, not only CP78 but also CP79 cells excreted pyruvate . Experiments using {1,4-14C}succinate as a carbon source revealed that pyruvate was formed by decarboxylation of one carboxyl group of succinate and that the pyruvate excretion amounted to about 40% of the total succinate degraded . Experiments designed to elucidate the mechanism of the excretion yielded the following observations . (i) The concentration of pyruvate in CP78 cells grown on the C4-dicarboxylic acids mentioned above was not significantly changed upon amino acid starvation . (ii) Guanosine 5'-diphosphate-3'-diphosphate exerted no effect on the activities of several enzymes thought to be involved in pyruvate-related metabolism . It is suggested firstly that the excretion was not due to some impairment in the biosynthetic pathway of a particular amino acid, but was due to the stringent control of central amphibolic metabolism, and secondly that no de novo protein synthesis was involved in the excretion.

J Biochem (Tokyo), 1981 Nov, 90(5), 1321 - 31
Regulation of Escherichia coli phosphoenolpyruvate carboxylase by multiple effectors in vivo . II . Kinetic studies with a reaction system containing physiological concentrations of ligands; Izui K et al.; In an attempt to clarify the kinetic properties of Escherichia coli phosphoenolpyruvate (PEP) carboxylase {EC 4.1.1.31} in vivo and to evaluate the physiological significance of the individual effectors, saturation curves were obtained for each ligand with reaction mixtures (pH 7.3) containing "physiological concentrations" of the other ligands in various combinations . As the "physiological concentrations" of ligands, which are defined as the concentrations of ligands found in the glucose-grown cells, the following values were employed: PEP, 0.2 mM; acetyl-CoA(CoA-SAc), 0.4 mM; fructose 1,6-bisphosphate(Fru-1,6-P2), 2.0 mM; GTP, 1.0 mM; L-aspartate, 1.0 mM; L-malate, 1.0 mM (Morikawa, M., Izui, K., Taguchi, M., & Katsuki, H . (1980) J . Biochem . 87, 441--449) . In the absence of any activator the enzyme activity was very low . CoASAc was the most powerful activator . The other two activators (Fru-1,6-P2 and GTP) exhibited essentially no activation alone, but produced a strong synergistic activation with CoASAc . The severe inhibition by L-aspartate or L-malate was effectively alleviated only through this synergistic action of the activators . The presence of all three activators decreased the half-saturation concentration (S0.5) of PEP from 15 mM to 0.35 mM and increased the maximal velocity attainable at infinite concentration of PEP about 15-fold . In the system containing all five effectors, which is close to the in vivo condition, the saturation curve of PEP was sigmoidal with a Hill coefficient of 1.6 and with an S0.5 value of 3.0 mM, which is about 15-fold larger than its "physiological concentration." On the basis of the rate-concentration curve for each effector obtained with the reaction mixture containing PEP and the other effectors at "physiological concentrations," it was suggested that all five effectors significantly contribute to the enzyme activity in vivo . Palmitoleate, another activator of the enzyme, showed no activation in such a reaction mixture . The sensitivity of the enzyme to the "physiological concentration" of each effector was also observed in an in situ system using permeabilized E . coli cells, where the enzyme concentration was as high as in vivo.

Am J Vet Res, 1981 Nov, 42(11), 1993 - 8
Assay of penetrability of bovine papillary duct implanted with Escherichia coli endotoxin; Schultze WD; A method has been developed to assay the relative penetrability to a solute of the papillary duct (streak canal) of the bovine teat . Escherichia coli lipopolysaccharide endotoxin (1.0 microgram in 2.5 microliter of sterile distilled water) was implanted at a depth of 3 mm from the distal end (papillary orifice) of the papillary duct . If penetration by the irritant occurred after any one of 5 successive daily implantations, the inflammatory response of the mammary gland was detected by a precipitous increase (by at least 15 mm) in the Wisconsin mastitis test score of quarter foremilk samples, and the test was scored as positive . Results of the penetrability assay were highly repeatable among quarters of certain cows . Cows differed as to the pattern of penetrability of their papillary ducts, with about half of 70 tested cows scoring 4 positive or 4 negative.

J Dairy Sci, 1981 Nov, 64(11), 2234 - 9
Endotoxin dose-related leukocytosis in milk of guinea pigs; McKenzie WN Jr et al.; Objectives were to determine minimum and maximum doses of Escherichia coli endotoxin that would stimulate leukocytosis in the mammary gland of the guinea pig . Endotoxin concentrations of .005, .05, .5, 5.0, and 50.0 microgram/ml in 1 ml were used to stimulate migration of polymorphonuclear leukocytes in milk of guinea pigs at five times (2, 4, 6, 8, and 12 h postinjection with endotoxin) . There were three animals in each experimental group per endotoxin concentration . Each animal served as its own control by having sterile saline injected into one mammary gland and endotoxin into the other . A control group of three animals had no saline or endotoxin injected . Guinea pigs were milked with a modified milking apparatus . Leukocyte numbers in the uninjected mammary gland did not differ from those in saline injected glands . The difference in mean leukocyte numbers between saline injected and .005 microgram/ml endotoxin injected glands approached significance at 5% . There was no difference between mean numbers recruited by the 5.0 and 50.0 microgram/ml endotoxin doses at 4, 6, 8, and 12 h . The chemotactic effect and, therefore, the rate of polymorphonuclear leukocyte recruitment was maximal at the 5 microgram/ml endotoxin dose.

Ann Microbiol (Paris), 1981 Nov-Dec, 132 B(3), 321 - 36
{Theoretical model of the predator-prey interaction kinetics between "Bdellovibrio bacteriovorus" and "escherichia coli" (author's transl)}; Marchand A et al.; A theoretical model is suggested in order to explain the main features of the interaction kinetics between the micropredator Bdellovibrio bacteriovorus and its prey Escherichia coli . Three parametes are used in this model: the incubation time T, the fixation rate constant k, and the predator multiplication factor a . Their values can be determined from the experimental variations of the total predator concentration p, and the total density of preys (c + c') . An experimental study of the predation kinetics was performed at various temperatures (25-40 degrees C) and in media with different Ca++ and Mg++ concentrations . From the values of parameters obtained, theoretical prey and predator density curves were computer-simulated; they agree satisfactorily with the experimental curves . The parameters values are quite reasonable and in good agreement with previous findings.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1981 Nov, 250(4), 470 - 7
An outbreak (?) caused by Escherichia coli O126:K71:H5 in premature infants; Goldhar J et al.; The strain E . coli O126:K71:H5 was recovered during a 6 week period, from stool samples of 13 premature infants hospitalized in the Premature Infants Nursery Unit . Of a total of 147 tested stool samples, 69 were positive . This strain was also recovered for a long period from the respiratory tract of one of the infants . The strain had a low virulence for the infants . No clear relationship could be demonstrated between clinical status and recovery of the strain . The strain did not possess any of the well defined pathogenic mechanisms, namely enterotoxins LT and ST, and invasiveness . The production of a thermolabile substance toxic for Vero cells was demonstrated . The strain possessed both guinea pig mannose-sensitive and human mannose resistant hemagglutinins . In spite of the production of thermolabile toxic substance for Vero cells and in spite of its contagiousness and intestinal tract colonization capacity, the strain displayed the characteristics of an opportunistic pathogen rather than classic EPEC.

Cell, 1981 Nov, 26(3 Pt 1), 305 - 14
The tyrT locus: termination and processing of a complex transcript; Rossi J et al.; The tyrT locus of E . coli contains a 208 bp spacer region that separates two copies of sequence encoding tRNATyr1 . The spacer includes a 120 bp sequence that is homologous to a sequence that is repeated three times in the distal portion of the tyrT locus . The tyrT locus possesses a graded set of transcription termination sites that are spaced at 180 base intervals, corresponding to the distal repeated gene structure . The major termination site occurs within the second repeat unit, 225 bases beyond the mature tRNA sequences . In the presence of a temperature-sensitive rho protein there is increased read-through at this site to a termination site located 180 bases downstream in the third repeat and to several termination sites even further downstream . The primary native transcript, in the region distal to the second tRNA, carries the information for a low molecular weight, extremely basic protein . Although analogous coding sequences are present in the spacer and other repeat units, because of single base substitutions these sequences are pseudogenes . The parallel between the tyrT and TyrU gene clusters is discussed in relation to dual function transcripts that specify both tRNA and protein.

Mol Biol (Mosk), 1981 Nov-Dec, 15(6), 1234 - 44
{Translation of RNA 4 from plant brome mosaic virus in an Escherichia coli cell-free system with pure protein factors in translation}; Belitsina NV et al.; Translation by RNA 4 from plant brome mosaic virus coding for the virus coat protein in an E . coli cell-free system with pure factors of translation has been studied . It has been shown that the initiation of translation by this mRNA depends completely on the three E . coli initiation factors . Optimal ionic conditions for the formation of the initiatory 70S times fMet-tRNA times RNA 4 complex have been found . It has been shown that this complex is stable in conditions of zonal centrifugation . On the basis of reaction with puromycin it has been determined that the initiatory fMet-tRNA in this complex occupies the donor-tRNA-binding site of the ribosome . By the competence of the initiatory ribosomal complex for binding with Ser-tRNA (serine is the N-terminal amino acid in the virus coat protein) it can be concluded that the ribosomal and the E . coli initiation factors recognize the initiatory codon of the RNA r from brome mosaic virus . Peptide synthesis induced by RNA 4 has been obtained on E . coli ribosomes with five pure factors of translation: IF-1, IF-2A, IF-3, EF-Tu or (Tu--Ts) and EF-G . The dependence of elongation on the Mg2+ concentration in the medium at RNA 4 translation has been determined.

J Virol, 1981 Nov, 40(2), 599 - 601
Coliphage 186 infection requires host initiation functions dnaA and dnaC; Hooper I et al.; We show that coliphage 186 infection is dependent upon host initiation functions, dnaA and dnaC, which differentiates the phage from lambda and P2 . The possibility is therefore entertained that the delay in 186 replication seen after infection of UV-irradiated bacterial cells reflects the temporary unavailability of one or both these functions . Infections with P1 and Mu need host dnaC but not dnaA and show some sensitivity to preirradiation of the host but are not as sensitive as 186.

J Virol, 1981 Nov, 40(2), 335 - 40
The transient inability of the conjugating female cell to host 186 infection explains the absence of zygotic induction for 186; Woods WH et al.; In an Hfr(186) X F- cross, the 186 prophage on the incoming male chromosome is not induced, despite the fact that prophage 186 can be induced by other means (W . H . Woods and J.B . Egan, J Virol . 14:1349-1356, 1974) . We show here that the conjugating female is temporarily inhibitory to infection by 186, and this delay, we postulate, enables cI repression to be reestablished before the female cell recovers its 186 sensitivity.

Eur J Biochem, 1981 Nov, 120(2), 379 - 87
The mechanism of tryptophan binding to tryptophan synthase from Escherichia coli; Lane AN et al.; The kinetics of the binding of L-tryptophan to the alpha 2 holo beta 2 complex of tryptophan synthase from Escherichia coli have been measured by rapid-mixing techniques under conditions where tryptophan release is mainly rate-determining in tryptophan synthesis . The dependence of the three observable rate processes on the concentration of L-tryptophan suggests a mechanism in which a rapid binding step is followed by two isomerizations . The effect of the substrate analogue indolepropanol phosphate on the kinetics of binding and synthesis from L-serine and indole supports a branched mechanism with an unproductive enzyme-ligand complex being the major species . The productive enzyme-ligand complex absorbs light at 473 nm but not at 500 nm . These observations, and binding studies with D-tryptophan, suggest that at least two alterative modes of binding of L-tryptophan exist on the enzyme . The effects of protons, indole and indolepropanol phosphate on the three rate processes explain the dependence of kcat on the three non-competitive ligands.

Can J Microbiol, 1981 Nov, 27(11), 1231 - 3
Mapping of a new pan mutation in Escherichia coli K-12; Manch JN; A previously unmapped pan gene was localized in an Escherichia coli K-12 strain using Hfr and F' matings and by transduction studies . This Pan- mutation was shown by supplementation studies to impart a block in pantoate biosynthesis . The pan gene cotransduced with rha, metB, and argE placing it at 87 min.

Proc Natl Acad Sci U S A, 1981 Nov, 78(11), 7033 - 7
Chi activity during transduction-associated recombination; Dower NA et al.; Chi is a genetic element that stimulates phage lambda recombination by the Escherichia coli recBC pathway during lytic infection {Stahl, F . W . (1979) Annu . Rev . Genet . 13, 7--24} . Herein we show that chi in lambda prophage influences exchange distribution in P1 phage-mediated transduction and in conjugation . This demonstration encourages the view that chi may influence genetic exchange in E . coli in the total absence of lambda.

Proc Natl Acad Sci U S A, 1981 Nov, 78(11), 7024 - 7
Alternate pathways of DNA replication: DNA polymerase I-dependent replication; Niwa O et al.; We have previously shown that some Escherichia coli {derivatives of strain HS432 (polA1, polB100, polC1026)} can replicate DNA at a restrictive temperature in the presence of a polCts mutation and that such revertants contain apparent DNA polymerase I activity . We demonstrate here that this strain of E . coli becomes temperature-resistant upon the introduction of a normal gene for DNA polymerase I or suppression of the polA1 nonsense mutation . Such temperature-resistant phenocopies become temperature-sensitive upon introduction of a temperature-sensitive DNA polymerase I gene . Our results confirm that DNA replication is DNA polymerase I-dependent in the temperature-resistant revertants, indicating that an alternative pathway of replication exists in E . coli . HS432 contains a transducible locus (which we term pcbA) that can support an alternate pathway in other E . coli strains, so the effect of suppression of polCts is a general one.

Proc Natl Acad Sci U S A, 1981 Nov, 78(11), 7010 - 4
Base-pair substitution hotspots in GAG and GCG nucleotide sequences in Escherichia coli K-12 induced by cis-diamminedichloroplatinum (II); Brouwer J et al.; Cell killing and mutation induction by cis- and trans-Pt(NH3)2Cl2 in Escherichia coli were examined by studying forward mutagenesis in the lacI gene in cells with different repair capacities . Survival experiments showed that repair-proficient cells were slightly more sensitive for the cis isomer than for the trans isomer, whereas repair-deficient RecA and UvrB cells were extremely sensitive only for the cis compound . cis-Pt(NH3)2Cl2 induced mutagenesis in both wild-type cells and RecA cells but not in UvrB cells; whereas no detectable mutagenesis was induced by treatment with the trans compound . Examination of the nature of the mutations induced by cis-Pt(NH3)2Cl2, by using the LacI system, revealed that base-pair substitutions leading to nonsense mutants are only induced in wild-type cells, suggesting that the intact products of both the uvrB and the recA gene are necessary for the repair responsible for this type of mutagenesis . Investigation of the nonsense mutants reveals that 70% of these mutations result from GC leads to TA or GC leads to AT substitutions at sites where the guanine is part of a GAG or GCG sequence . These results are discussed in relation to existing theories on the interaction between Pt compounds and DNA . A model for Pt--DNA adducts, leading to base-pair substitutions, is proposed.

Proc Natl Acad Sci U S A, 1981 Nov, 78(11), 6826 - 30
Cloning and sequence of cDNA coding for alpha 1-antitrypsin; Kurachi K et al.; Recombinant plasmids containing human and baboon cDNA have been screened for alpha 1-antitrypsin, a major serine protease inhibitor present in blood . One plasmid, designated pBa alpha 1a2, was found to contain a cDNA insert of 1352 base pairs coding for the baboon inhibitor . It included 45 nucleotides that code for 15 amino acids present in the amino-terminal signal sequence of the protein, 1182 nucleotides that code for 394 amino acids in the mature protein, a stop codon, and a noncoding region of 76 nucleotides . Comparison of the amino acid sequences of baboon alpha 1-antitrypsin, human antithrombin III, and chicken ovalbumin indicated that these three proteins are about 230% homologous . A second plasmid, designated pH alpha 1a1, was found to contain a human cDNA insert of 306 base pairs . This plasmid coded for 69 amino acids present in the carboxyl-terminal region of human alpha 1-antitrypsin . The human and baboon cDNAs and their amino acid sequences are greater than 96% homologous.

Proc Natl Acad Sci U S A, 1981 Nov, 78(11), 6759 - 63
Changes in the hydrogen exchange kinetics of Escherichia coli aspartate transcarbamylase produced by effector binding and subunit association; Lennick M et al.; Large changes in solvent accessibility to aspartate transcarbamylase (aspartate carbamoyltransferase, carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2), as monitored by tritium exchange, result from binding of substrates and substrate analogs to the catalytic subunit (c3), binding of nucleoside triphosphates to the regulatory subunit (r2), and subunit association . Rates of exchange are reduced in each of these cases, although to different degrees . Succinate, in the presence of carbamoyl phosphate, retards exchange from c3 no more than carbamoyl phosphate alone, and less than N-phosphonacetyl-L-aspartate, a bisubstrate analog . Larger changes in rates of exchange from r2 are produced by CTP than by ATP; however, both CTP and ATP accelerate exchange from c3 to the same extent . The changes in the kinetics of exchange that result from binding of both substrate analogs and nucleoside triphosphates to the native enzyme (c6r6) are much smaller . Carbamoyl phosphate, with or without succinate, retards exchange only slightly, while the bisubstrate analog has a somewhat larger effect . Experiments with reconstituted enzyme, in which only c3 is tritium labeled, indicate that changes in solvent accessibility produced by active site ligands are largely confined to c3 . Neither CTP nor ATP alters the overall rate of exchange from c6r6 significantly . The possibility of opposing changes in the two types of subunits was ruled out in experiments in which only one subunit was labeled . The nonadditive effects of ligation and subunit association imply a set of responsive protons common to both processes and suggest that they are linked not only thermodynamically and functionally but also dynamically.

Proc Natl Acad Sci U S A, 1981 Nov, 78(11), 6652 - 6
Arrangement of the subunits in the ribosome of Escherichia coli: demonstration by immunoelectron microscopy; Kastner B et al.; The three-dimensional locations of Escherichia coli ribosomal proteins S13, L1, and L7/L12 on the surface of ribosomal subunits and 70S monomeric ribosomes were determined by electron microscopy of antibody-labeled ribosomal particles . A new approach to orient the subunits within 70S ribosomes was developed that used 30S.70S.50S triples that were prepared by simultaneous combination with one antibody directed against a 30S protein and another directed against a 50S protein . Electron microscope studies of triples obtained with the antibody combinations anti-S13/anti-L1 and anti-S13/anti-L7/L12 showed that, in 70S monomeric ribosomes, the head of the 30S subunit is proximate to protein L1 and the peptidyl transferase center but far from the rod-like appendage containing proteins L7 and L12.

J Med Microbiol, 1981 Nov, 14(4), 381 - 9
Escherichia coli antibodies in opsonisation and protection against infection; van Dijk WC et al.; The opsonic and protective capacities of rabbit antisera against Escherichia coli O, K and core-glycolipid cell-wall antigens were compared with specific antibody titres as measured by agglutination and enzyme-linked immunosorbent assay . Anti-O antisera were opsonic and protective against two noncapsulate strains . Only anti-K antisera were opsonic and protective against a K-antigen-containing strain . In a mouse model anti-core-glycolipid antiserum was not protective against challenge even by a strain bearing only core glycolipid.

J Clin Microbiol, 1981 Nov, 14(5), 473 - 8
Single radial immune hemolysis test for detection of Escherichia coli thermolabile enterotoxin; Serafim MB et al.; A single radial immune hemolysis test for the detection of thermolabile enterotoxin has been developed for routine purposes . Stationary cultures from enterotoxigenic Escherichia coli in Casamino Acids-yeast extract medium may be used for the detection of this enterotoxin, and under the conditions of the experiment, the single radial immune hemolysis test was as sensitive as the passive immune hemolysis test . The results obtained in the single radial immune hemolysis test agreed entirely with those obtained in the passive immune hemolysis test, and no false-positive reactions were obtained when cholera antitoxin diluted 1:80 was used . The assay is easy to perform, inexpensive, and specially designed for less-equipped laboratories.

Infect Immun, 1981 Nov, 34(2), 637 - 9
Protection in rats immunized with Escherichia coli heat-stable enterotoxin; Klipstein FA et al.; Rats immunized with a semipurified preparation of the Escherichia coli heat-stable (ST) enterotoxin conjugated with a protein carrier were protected against challenge with semipurified or purified ST and viable organisms of multiple heterologous serotypes that produce only ST (LT-/ST+), but they were not protected against heal-labile (LT) toxin or viable strains which produce LT either alone (LT+/ST-) or together with ST (LT+/ST+).

Surgery, 1981 Nov, 90(5), 853 - 9
Skeletal muscle insulin resistance during Escherichia coli bacteremic shock in the dog; Raymond RM et al.; Skeletal muscle glucose uptake during close, intra-arterial insulin infusion was studied before and during live Escherichia coli bacteremic shock in the dog . An in vivo, constant-flow perfused gracilis muscle preparation was used . Insulin infusion before shock resulted in a 395% increase in muscle glucose uptake, which was independent of changes in muscle lactate production or oxygen uptake . At 1, 2, and 3 hours of shock, insulin infusion had no effect on gracilis muscle glucose uptake . This loss of responsiveness to insulin occurred with no change in muscle oxygen uptake, muscle venous PO2, or muscle blood flow (held constant) . On the other hand, during nonshock control experiments, muscle glucose uptake in response to insulin infusion was maintained during the 3-hour protocol . These data demonstrate that skeletal muscle insulin resistance develops early during bacteremic shock.

Lab Invest, 1981 Nov, 45(5), 435 - 41
Vascular responses during acute neutrophilic inflammation . Their relationship to in vivo neutrophil emigration; Issekutz AC; Hyperemia, an increase in vascular permeability, and the emigration of leukocytes are the basic manifestations of the acute inflammatory reaction . Many previous studies have employed phagocytosable material to induce inflammation and neutrophil infiltration . In this study, we induced neutrophil infiltration into rabbit skin by injecting the soluble chemotactic stimuli, zymosan-activated plasma, C5adesArg, N-formyl-methionyl-leucyl-phenylalanine, and factors released by Escherichia coli . During the evolution of the response, 51Cr-labeled leukocytes were used to quantitate neutrophil influx, 125I-labeled albumin was used to quantitate permeability, and 86Rb was used to quantitate blood flow . Simultaneous measurements showed a close correlation in time between the degree and peak rate of neutrophil influx and the degree of hyperpermeability and hyperemia . Dose-response experiments performed with 2 to 90 per cent (v/v) zymosan-activated plasma showed a direct correlation between the rate of neutrophil influx and the degree of vascular permeability in blood flow . No vascular responses were induced after injection of these chemotactic stimuli with neutrophil infiltration were inhibited by indomethacin or aspirin . It is concluded that the vascular hyperpermeability and hypermia accompanying neutrophilic inflammatory reactions may be induced by neutrophils during their migration across the microvascular walls and that prostaglandins may in part mediate these responses . Furthermore, phagocytosis by the neutrophils is not required to induce these vascular changes.

J Bacteriol, 1981 Nov, 148(2), 730 - 3
Bilinear cell growth of Escherichia coli; Kubitschek HE; Recent electron micrograph measurements of bacterial dimensions in exponentially growing cultures of Escherichia coli support a model of bilinear increase in cell surface area and volume, with a sharp doubling in growth rate at a discrete age during the cell cycle . The results also indicate coordinate regulation of increase of surface area and volume.

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