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 partitionin