Prokaryotes

Nature, 1986 May 22-28, 321(6068), 449 - 50
Sequence-directed curvature of DNA; Hagerman PJ; DNAs from both prokaryotic and eukaryotic organisms have yielded restriction fragments which manifest markedly anomalous electrophoretic behaviour (reduced mobility) when run on polyacrylamide gels . We have shown previously that the abnormal electrophoretic behaviour of one such fragment is a consequence of stable curvature of the helix axis in solution . The molecules involved tend to contain oligo(dA)-oligo(dT) runs which are approximately in-phase with the helix repeat; however, the precise structural elements responsible for DNA curvature have not been identified . One popular model for curvature invokes a non-coplanar 'wedge-like' conformation of ApA/TpT dinucleotide pairs . Despite a lack of direct evidence in support of this model, it has been used to provide quantitative estimates of curvature . To critically evaluate the ApA wedge model, we have performed an electrophoretic analysis of a series of closely related DNA polymers in which oligo(dA)-oligo(dT) runs of different polarity were compared . We conclude that ApA dinucleotide wedges cannot account for DNA curvature . Therefore, quantitative estimates for ApA wedge deformations, based solely on apparent curvature, cannot be correct.

Nature, 1986 May 22-28, 321(6068), 441 - 3
Specific inhibition of herpesvirus ribonucleotide reductase by a nonapeptide derived from the carboxy terminus of subunit 2; Cohen EA et al.; Ribonucleotide reductase, an essential enzyme for the synthesis of deoxyribonucleotides, is formed by the association of two nonidentical subunits in almost all prokaryotic and eukaryotic cells . The same model probably holds for the herpes simplex virus (HSV)-encoded ribonucleotide reductase; two polypeptides of relative molecular mass 136,000 (136K; H1) and 40K (H2) (referred to elsewhere as RR1 and RR2; see for example, Dutia et al.) have been associated with the viral enzyme by both genetic and immunological studies . Furthermore, DNA sequence analyses have shown significant stretches of amino-acid homology between these viral polypeptides and those of, respectively, subunit 1 (ref . 12) and subunit 2 (ref . 13) of the Escherichia coli and mammalian enzymes . To assess the involvement of the 40K polypeptide in reductase activity, we synthesized a nonapeptide corresponding to the sequence of its carboxy terminus with the intention of raising neutralizing antibodies specific for the viral activity (E.A.C . et al., in preparation) . We report here the unexpected finding that the nonapeptide itself specifically inhibits the HSV ribonucleotide reductase activity in a reversible, non-competitive manner, and we suggest that it does this by impairment of the correct association of the two subunits . This phenomenon emphasizes the potential usefulness of synthetic peptides in probing critical sites involved in macromolecular interactions.

Nature, 1986 May 22-28, 321(6068), 439 - 41
Specific inhibition of herpesvirus ribonucleotide reductase by synthetic peptides; Dutia BM et al.; Ribonucleotide reductase is an essential enzyme for DNA synthesis in all prokaryotic and eukaryotic cells; it catalyses the reductive conversion of ribonucleotides to deoxyribonucleotides . Several herpesviruses including herpes simplex virus type 1 (HSV-1), HSV-2, pseudorabies virus (PRV), equine herpesvirus type 1 (EHV-1) and Epstein-Barr virus (EBV) have been found to induce novel ribonucleotide reductase activities . There is evidence that the HSV-1 ribonucleotide reductase activity is virus-encoded and essential for virus replication . This makes herpesvirus ribonucleotide reductases potential targets for antiviral chemotherapy . The HSV-1-encoded enzyme consists of two subunits: V136, the large subunit of relative molecular mass (Mr) 136,000 (136K) (RR1), which has been shown to be essential for enzyme activity, and V38, the small subunit (RR2) which forms a complex with the large subunit and is also likely to be essential for enzyme activity . Two particular features of the enzyme make it an attractive antiviral target . First, there is evidence for a common, highly conserved herpesvirus ribonucleotide reductase and second, the interaction between the large and small subunits may itself be exploitable . Here we identify a synthetic peptide which specifically inhibits the activity of virus-induced enzyme . We deduce that the mechanism of inhibition involves interference with the normal interaction between the two types of subunit.

Biochemistry, 1986 May 20, 25(10), 2765 - 9
Proteins from the prokaryotic nucleoid: 1H NMR study of the quaternary structure of Escherichia coli DNA binding protein NS (HU); Paci M et al.; The quaternary interactions of Escherichia coli DNA binding proteins NS1, NS2, and NS (NS1 + NS2) have been studied by 1H NMR spectroscopy at 400 MHz following the reversible spectral changes produced by temperature increases on the resonances (Phe ring and His C-2 protons) whose spectral characteristics reflect the formation and dissociation of either homologous or heterologous interactions . These changes include (a) a progressive intensity decrease of the Phe resonances shifted to high field by stacking interactions, (b) a progressive intensity increase of the resonances due to freely rotating Phe, and (c) splitting of the His C-2 proton resonance . The association constants and thermodynamic parameters for the homologous and heterologous interactions were calculated from the molar fractions of the relevant molecular species by assuming that the above effects are due to the existence of simple association equilibria . It was found that two (out of three) phenylalanine residues of each polypeptide chain are involved in quaternary interactions . Quantitative data concerning the internal mobility and mutual orientations in aggregates of these Phe rings were also obtained . From the calculated association constants, from comparison of these data with recent protein-protein cross-linking results {Losso, M . A., Pawlik, R . T., Canonaco, M . A., & Gualerzi, C . O . (1986) Eur . J . Biochem . 155, 27-32}, and from other considerations, we suggest that even though stacking of the Phe rings occurs at the interface between monomers, the temperature-dependent alteration of the Phe spectrum monitors shifts of the dimer in equilibrium tetramer equilibrium whereas the splitting of the His C-2 proton resonance most likely monitors the equilibrium between tetramers and larger aggregates.

J Theor Biol, 1986 May 7, 120(1), 85 - 98
Eukaryotic mRNA 5'-leader sequences have dual regions of complementarity to the 3'-terminus of 18s rRNA; Maroun LE et al.; We have used a microcomputer program to test eukaryotic mRNA 5'-leader sequences for complementarity to the 3'-terminus of 18S rRNA . No mismatched bases, bulge loops, or viral mRNA's were utilized . At least one-fourth of the more than 200 mRNA's studied were found to have two distinct regions of complementarity which resulted in an ability to bind to two separate rRNA regions (GAAGG and UUUGG) . The analysis of 60 mRNAs with these dual sites resulted in a consensus structure that was a mean distance of 11.75 bases 5' from the initiator AUG and had an average predicted interstrand binding strength of delta G = -13.40 kcal . These characteristics compare favorably to those observed for the prokaryotic 16S rRNA-mRNA Shine and Dalgarno bond.

J Mol Biol, 1986 May 5, 189(1), 13 - 24
Expression of the prokaryotic gene for chloramphenicol acetyl transferase in Drosophila under the control of larval serum protein 1 gene promoters; Davies JA et al.; We have linked the protein coding region of the prokaryotic gene for chloramphenicol acetyl transferase (CAT) to the promoter region of the Drosophila genes for larval serum protein 1 (LSP1) . These regions consist of 1.65 X 10(3) and 2.25 X 10(3) base long DNA segments upstream from the LSP1 alpha and beta genes, respectively . The hybrid genes have been inserted into a P-element transformation vector and the constructs introduced into cultured Drosophila Kc cells by calcium phosphate-mediated transfection, or into the germ-lines of flies by P-element-mediated transformation . CAT expression occurs in approximately 1% of the cultured cells following transfection and the accumulation of protein is maximal three to four days after transfection; and it is dependent upon the presence of the LSP1 sequences . We have also obtained several lines of transformed flies that carry the LSP1-CAT genes in their germ-line . The onset of synthesis of functional chloramphenicol acetyl transferase in these organisms occurs in the late third larval instar, rising to a maximum at puparium formation . We continue to detect CAT until the first few hours of adulthood . Assays of CAT activity in the homogenates of dissected tissues indicated that the genes are only expressed in the fat body, as are the endogenous LSP1 genes . We have confirmed this tissue-specific localization of CAT in indirect immunofluorescence using an anti-CAT monoclonal antibody . Northern blots indicate that CAT transcripts are found only in the fat body but their abundance is an order of magnitude lower than endogenous LSP1 transcripts . Primer extension experiments show that transcription of the hybrid genes is initiated at the same nucleotide as the endogenous LSP1 genes . Taken together these data indicate that the 1.65 X 10(3) and 2.25 X 10(3) base segments of DNA upstream from the LSP1 alpha and LSP1 beta genes contain cis-acting regulatory elements necessary for correct tissue and temporal specificity of LSP1 gene expression.

Proc Natl Acad Sci U S A, 1986 May, 83(10), 3141 - 5
Homology between RNA polymerases of poxviruses, prokaryotes, and eukaryotes: nucleotide sequence and transcriptional analysis of vaccinia virus genes encoding 147-kDa and 22-kDa subunits; Broyles SS et al.; We have determined the nucleotide sequence of a region of the vaccinia virus genome encoding RNA polymerase subunits of 22 and 147 kDa and have mapped the 5' and 3' ends of the two mRNAs . The predicted amino acid sequence of the vaccinia 147-kDa subunit shows extensive homology with the largest subunit of Escherichia coli RNA polymerase, yeast RNA polymerases II and III, and Drosophila RNA polymerase II . The regions of homology between the five RNA polymerases are subdivided into five separate domains that span most of the length of each . A sixth domain shared by the vaccinia and the eukaryotic polymerases is absent from the E . coli sequence . In all specified regions, the vaccinia large subunit has greater homology with eukaryotic RNA polymerases II and III than with the E . coli polymerase . Vaccinia virus and eukaryotic RNA polymerases may therefore have evolved from a common ancestral gene after the latter diverged from prokaryotes.

EMBO J, 1986 May, 5(5), 1049 - 56
Two tandemly linked identical genes code for the glycosomal glyceraldehyde-phosphate dehydrogenase in Trypanosoma brucei; Michels PA et al.; Trypanosoma brucei contains two isoenzymes for glyceraldehyde-phosphate dehydrogenase (GAPDH); one enzyme resides in a microbody-like organelle, the glycosome, the other one is found in the cytosol . We show here that the glycosomal enzyme is encoded by two tandemly linked genes of identical sequence . These genes code for a protein of 358 amino acids, with a mol . wt of 38.9 kd . This is considerably larger than all other GAPDH proteins studied so far, including the enzyme that is located in the cytosol of the trypanosome . The glycosomal enzyme shows 52-57% homology with known sequences of GAPDH proteins from 10 other organisms, both prokaryotes and eukaryotes . The residues that are involved in NAD+ binding, catalysis and subunit contacts are well conserved between all these GAPDH molecules, including the trypanosomal one . However, the glycosomal protein of T . brucei has some distinct features . Firstly, it contains a number of insertions, 1-8 amino acids long, which are responsible for the high mol . wt of the protein . Secondly, an unusually high number of positively charged amino acids confer a high isoelectric point (pI 9.3) to the protein . Part of the additional basic residues are present in the insertions . We discuss the genomic organization of the genes for the glycosomal GAPDH and the possibility that the particular features of the protein are involved in its transfer from the cytoplasm, where it is synthesized, into the glycosome.

Nature, 1986 Apr 24-30, 320(6064), 766 - 8
Homoeo-domain homology in yeast MAT alpha 2 is essential for repressor activity; Porter SD et al.; The MAT alpha locus of the yeast Saccharomyces cerevisiae encodes two regulatory proteins, alpha 1 and alpha 2, which are responsible for determining the alpha-cell type . MAT alpha 1 is a positive regulator of alpha-cell-type-specific genes, and MAT alpha 2 is a negative regulator of a-cell-type-specific genes . MAT alpha 2 also determines the a/alpha diploid cell type, in conjunction with the MATa product, a1, by repressing haploid cell-type-specific genes . The MAT alpha 2-encoded protein binds specifically in vitro to a DNA sequence found upstream of several a-specific genes and is thus thought to exert its control directly at the transcriptional level of target genes . In an initial attempt to understand the molecular basis of the interaction of alpha 2 with DNA, we have saturated with missense mutations the segment of alpha 2 that is weakly homologous to a conserved prokaryote DNA-binding structure and to a portion of the higher eukaryote homoeo domain to ascertain the possible functional significance of this homology in alpha 2 . We report here that most of the amino-acid residues in alpha 2 which correspond to conserved amino acids in the prokaryote DNA-binding proteins and in the homoeo domain are essential for the two repressor activities of alpha 2, that is, the repression of a-specific genes and of haploid-specific genes . Mutations in a subset of these amino-acid residues more severely affect the ability to repress a-specific genes than haploid-specific genes.

Biochemistry, 1986 Apr 22, 25(8), 2212 - 20
Mechanism of action of a mammalian DNA repair endonuclease; Doetsch PW et al.; The mechanism of action of a DNA repair endonuclease isolated from calf thymus was determined . The calf thymus endonuclease possesses a substrate specificity nearly identical with that of Escherichia coli endonuclease III following DNA damage by high doses of UV light, osmium tetroxide, and other oxidizing agents . The calf thymus enzyme incises damaged DNA at sites of pyrimidines . A cytosine photoproduct was found to be the primary monobasic UV adduct . The calf thymus endonuclease and E . coli endonuclease III were found to possess similar, but not identical, DNA incision mechanisms . The mechanism of action of the calf thymus endonuclease was deduced by analysis of the 3' and 5' termini of the enzyme-generated DNA scission products with DNA sequencing methodologies and HPLC analysis of the material released by the enzyme following DNA damage . The calf thymus endonuclease removes UV light and osmium tetroxide damaged bases via an N-glycosylase activity followed by a 3' apurinic/apyrimidinic (AP) endonuclease activity . The calf thymus endonuclease also possesses a novel 5' AP endonuclease activity not possessed by endonuclease III . The product of this three-step mechanism is a nucleoside-free site flanked by 3'-and 5'-terminal phosphate groups . These results indicate the conservation of both substrate specificity and mechanism of action in the enzymatic removal of oxidative base damage between prokaryotes and eukaryotes . We propose the name redoxy endonucleases for this group of enzymes.

J Biol Chem, 1986 Apr 15, 261(11), 4781 - 4
Chloroplast initiation factor 3 from Euglena gracilis . Identification and initial characterization; Kraus BL et al.; A chloroplast ribosome dissociation factor (IF-3chl) has been identified in whole cell extracts of Euglena gracilis . This work represents the first report of an organellar ribosome dissociation factor . E . gracilis IF-3chl facilitates the dissociation of Escherichia coli ribosomes as demonstrated by sucrose density gradient analysis . Chloroplast IF-3 stimulates initiation complex formation on E . coli ribosomes with natural mRNA from the bacteriophage MS2 . In addition, IF-3chl is effective in initiation complex formation with Euglena chloroplast or E . coli ribosomes in the presence of synthetic mRNA . IF-3chl is induced 12-fold by exposure of the cells to light . The chloroplast factor has been purified 30-fold by chromatography on DEAE-cellulose and phosphocellulose . The chromatographic properties of this factor differ considerably from those of prokaryotic ribosome dissociation factors.

Biochem J, 1986 Apr 1, 235(1), 25 - 31
Fluxes through the prokaryotic and eukaryotic pathways of lipid synthesis in the '16:3' plant Arabidopsis thaliana; Browse J et al.; The kinetics of {1-14C}acetate incorporation in Arabidopsis thaliana L . (Heyn) showed almost equal labelling of phosphatidylcholine (PC) and diacylgalactosylglycerol (DGG) at early times and the transfer of radioactivity from PC to DGG and diacyldigalactosylglycerol (DDG) at longer times . These kinetics demonstrated the parallel operation of the prokaryotic and eukaryotic pathways of lipid synthesis {Roughan & Slack (1982) Annu . Rev . Plant Physiol . 33, 97-132} in this tissue . At 2 h after the application of {1-14C}acetate, more than 85% of the radioactivity at the sn-2 position of each chloroplast lipid was in 16-carbon fatty acids . However, after 60 h, molecular species containing labelled C18 fatty acids at position sn-2 and presumably derived from microsomal PC made a large contribution (20-70%) to each chloroplast lipid except phosphatidylglycerol . These findings are consistent with the contention that the chain length of the fatty acid at the sn-2 position of glycerol is an accurate predictor of whether a particular lipid molecule has been synthesized by the prokaryotic or eukaryotic pathway . At 30 min after the start of {1-14C}acetate labelling, only 12.3% of the radioactivity in PC was in saturated fatty acids, but the proportion increased steadily to 24.3% after 142 h . It is suggested that steps involved in the conversion of PC to chloroplast lipids on the eukaryotic pathway discriminate against palmitate-containing species . The step involved does not appear to be transfer of PC to the chloroplast because extrachloroplastic and chloroplast membranes purified from Arabidopsis mesophyll protoplasts each contained PC with a fatty acid composition similar to that of the same lipid from leaves . Positional analysis of unlabelled lipids, together with the information summarized above, is used to construct a quantitative scheme of the fluxes through the prokaryotic and eukaryotic pathways during lipid synthesis in Arabidopsis . This scheme shows that 38% of the fatty acids synthesized de novo in the chloroplast enter the prokaryotic pathway in the chloroplast envelope . Of the 62% which are exported as acyl-CoA species to enter the eukaryotic pathway, 56% (34% of the total) are returned to complete synthesis of the chloroplast's complement of glycerolipids.

Proc Natl Acad Sci U S A, 1986 Apr, 83(7), 2133 - 7
Origin of eukaryotic introns: a hypothesis, based on codon distribution statistics in genes, and its implications; Senapathy P; A hypothesis for the origin of introns in eukaryotic genes is developed . By computer simulation it was found that the reading-frame lengths in a random nucleotide sequence are distributed in a negative exponential manner and that there exists an upper limit of about 200 codons in the length of the reading frames (RFs) . These characteristics suggest that, if primordial DNA contained a random nucleotide sequence, the most primitive cells would have been under selective pressure to eliminate interfering stop codons in order to increase the length of RFs . Further, they indicate that the only possible way that a coding sequence that is considerably longer than 600 nucleotides could be derived from the short coding sequences occurring in a random sequence would be to splice the short coding sequences and to eliminate the stretches of sequences containing clusters of inframe stop codons . Thus, introns are suggested to be those stretches of sequences containing interfering stop codons that were originally earmarked in the first primitive cells to be eliminated in order to enable the coding for long polypeptides . Because the statistical characteristics of codon distributions in today's eukaryotic DNA sequences resemble closely those of a random sequence and because the upper limit in the length of RFs (200 codons) in a random sequence corresponds precisely to the observed maximum length of exons in today's eukaryotic genes (600 nucleotides), it is suggested that introns originated in the most primitive unicellular eukaryotes when they evolved from primordial sequences . The data from the prokaryotic gene sequences indicate that prokaryotic genes may have been derived originally from primitive unicellular eukaryotic genes by losing introns from them.

Comput Appl Biosci, 1986 Apr, 2(1), 13 - 7
IBM microcomputer programs that analyze DNA sequences for tRNA genes; Shortridge RD et al.; A set of four computer programs that search DNA sequence data files for transfer RNA genes have been written in IBM (Microsoft) BASIC for the IBM personal computer . These programs locate and plot predicted secondary structures of tRNA genes in the cloverleaf conformation . The set of programs are applicable to eukaryotic tRNA genes, including those containing intervening sequences, and to prokaryotic and mitochondrial tRNA genes . In addition, two of the programs search up to 150 residues downstream of tRNA gene sequences for possible eukaryotic RNA polymerase III termination sites comprised of at least four consecutive T residues . Molecular biologists studying a variety of gene sequence and flanking regions can use these programs to search for the additional presence of tRNA genes . Furthermore, investigators studying tRNA gene structure-to-function relationships would not need to do extensive restriction mapping to locate tRNA gene sequences within their cloned DNA fragments.

Biochimie, 1986 Apr, 68(4), 505 - 15
Prokaryotic gene expression in vitro: transcription-translation coupled systems; Cenatiempo Y; Transcription-translation coupled systems have been developed to study prokaryotic gene expression . Several types of expression system have been described . The original system consists of a crude unfractionated Escherichia coli extract, which supports protein synthesis directed by a template DNA . Control of gene expression at the transcriptional stage has been studied using this unfractionated system . In this respect, two examples of particular interest, lactose and tryptophan operons, are described . Other systems are either partially reconstituted or highly defined, containing up to 30 purified factors necessary for transcription (RNA polymerase) and translation (aminoacyl-tRNA synthetases, initiation, elongation and release factors) . Additional differences between the various systems relate to the analysis of the gene products . Whereas most methods involve analysis of the totally synthesized protein, a particular system implies the formation of only the N-terminal di- or tripeptide of the gene product . Reconstituted systems have proved useful in studies on transcriptional, e.g., discovery and role of L factor, as well as translational regulation of gene expression, e.g., autogenous control of ribosomal protein synthesis.

Biochem Cell Biol, 1986 Apr, 64(4), 277 - 89
Enhancer-facilitated expression of prokaryotic and eukaryotic genes using human histone gene 5' regulatory sequences; Marashi F et al.; We examined the structural and functional properties of a human H3 histone gene promoter . The complete nucleotide sequence of an H3 structural gene and 515 nucleotides of 5' and 100 nucleotides of 3' flanking sequences were determined . The upstream region of this cell cycle dependent H3 histone gene, designated pST519, contains consensus sequences typical of genes transcribed by RNA polymerase II . To address promoter function directly, we determined the capability of the 5' flanking sequences to direct the transcription of two genes which are not functionally or structurally related . Fusion genes were constructed using the 5' flanking sequences of this human H3 histone gene and either human beta-globin or bacterial chloramphenicol acetyltransferase (CAT) coding sequences . Both of these fusion genes were expressed when transfected into HeLa cells . Under control of the pST519 histone gene promoter, a beta-globin mRNA transcript was initiated at the appropriate H3 (bp) enhancer, inserted upstream from the histone promoter in both fusion constructs, increased levels of beta-globin and CAT expression . Expression of the pST519 H3 histone gene in COS cells in the absence of the SV40 72-bp enhancer confirmed that the sequences required for promoting transcription reside within the 750-bp 5' flanking sequences and that the exogenous enhancer facilitates, but is not a prerequisite for, transcription . Enhancer-facilitated expression of a cell cycle dependent human H4 histone gene was also observed following transfection into mouse L cells and indicates that the regulatory sequences of human histone genes and transcription factors of mouse cells are compatible.

Eur J Biochem, 1986 Apr 1, 156(1), 85 - 94
Selective acylation of membrane proteins in Acholeplasma laidlawii; Nystrom S et al.; In membranes of the cell-wall-less prokaryote Acholeplasma laidlawii most proteins are of the integral type . A substantial fraction of these proteins are enriched in hydrophilic amino acid residues . Approximately 20 different major as well as minor proteins were found to be covalently modified with acyl chains . The same set of proteins are acylated when cells are grown in different fatty-acid-supplemented media . In individual proteins the ratio of palmitoyl/oleoyl acyl chains was 12-14 times larger than the acyl chain ratio in polar membrane lipids . The transmembrane protein D12 has close to two acyl chains per molecule . Proteins T2 and T4a, localized in the outer and inner leaflet of the membrane, respectively, occur each as pairs with a difference in relative molecular mass within each pair of approximately 2000 . Each of these proteins as well as the other acyl proteins, except the light form of T4a, has close to one acyl chain per molecule . The extent of acylation was increased for certain proteins and decreased for others by treatment with globomycin or phenethylalcohol . The relative amounts of the T2 and T4a pairs were affected by these drugs . It is concluded that the mechanism of acylation is different from that in Escherichia coli lipoprotein and Bacillus penicillinase . The mean hydrophobicity {Kyte & Doolittle (1982) J . Mol . Biol . 157, 105-132} of the A . laidlawii acyl proteins are similar to those of other bacterial acyl proteins but significantly lower than for non-acylated integral membrane proteins, supporting an anchoring function of the acyl chains . The number of membrane acyl proteins in A . laidlawii and two other mycoplasmas are at least twice that in other bacteria.

Science, 1986 Mar 28, 231(4745), 1553 - 5
Identification of HTLV-III/LAV sor gene product and detection of antibodies in human sera; Kan NC et al.; The nucleotide sequence of the genome of HTLV-III, the infectious agent etiologically associated with the acquired immune deficiency syndrome, predicts a small open reading frame, termed sor, located between the pol and env genes . A DNA segment containing 82 percent of the sor region was inserted into a prokaryotic expression vector, pJL6, to determine whether sor encodes a viral protein and to gain some insight into its possible function . The bacterially synthesized sor protein reacted with sera from individuals infected with HTLV-III, indicating that sor is expressed as a protein product or products that are immunogenic in vivo . Antibodies to the purified, bacterially synthesized sor protein were found to react specifically with the same protein and also with a protein of molecular weight 23,000 (23K) in HTLV-III-infected H9 cell extracts . The 23K protein comigrated with a protein immunoprecipitated by the serum of a hemophiliac patient with antibodies to HTLV-III, suggesting that this protein is probably the sor gene product.

Biochim Biophys Acta, 1986 Mar 26, 866(2-3), 109 - 19
Sequence signals in eukaryotic upstream regions; Nussinov R et al.; In eukaryotes, as in prokaryotes, evidence is accumulating showing that transcription factors recognize and bind to certain promoter elements . Sequence and chromatin structure perturbation specify the transcription initiation site and govern its efficiency . Two oligomers have been implicated in these processes: the TATAAATA and CCAAT . In the present work, all mammalian, non-mammalian vertebrate and invertebrate sequences accumulated in the database have been aligned by their mRNA start positions and scanned for recurrences of the 32 complementary triplets . The more significant signals are summarized here . In particular, TAT/ATA recurs very frequently further upstream, at -275, in addition to the 'classical' -40 position . Downstream their level is very low . The CAAT box complementary triplet components are not among the more striking signals . Closer examination of the -275 region indicates that to a large extent the signal is due to both the ATAT and the TATA quartets . Comparison of the frequencies of these quartets at -275 with the CAAT quartet at -80 suggests that the former signal is twice the strength of the latter . The oligomers' distribution charts support notions that several components are involved in recognition, making the regulatory regions more robust and less sensitive to mutations.

Nature, 1986 Mar 20-26, 320(6059), 287 - 8
Eukaryotic ribosomes that lack a 5.8S RNA; Vossbrinck CR et al.; The 5.8S ribosomal RNA is believed to be a universal eukaryotic characteristic . It has no (size) counterpart among the prokaryotes, although its sequence is homologous with the first 150 or so nucleotides of the prokaryotic large subunit (23S) ribosomal RNA . We report here an exception to this rule . The microsporidian Vairimorpha necatrix is a eukaryote that has no 5.8S rRNA . As in the prokaryotes, it has a single large subunit rRNA, whose 5' region corresponds to the 5.8S rRNA.

Eur J Biochem, 1986 Mar 17, 155(3), 475 - 83
Genes coding for the elongation factor EF-1 alpha in Artemia; Lenstra JA et al.; The elongation factor EF-1 alpha is one of the most abundant proteins in eukaryotic cells, where it catalyzes the binding of aminoacyl-tRNA to ribosomes . The genes coding for this protein in the brine shrimp Artemia were analyzed by gene cloning, electron microscopy and chromosomal blot hybridization . There are only a few (about four) copies of one type of gene per haploid genome . These genes contain five exons divided over 10(4) base pairs . Local rearrangements give rise to a number of gene variants . Cross-hybridizations of Artemia cDNA probes with yeast and Drosophila DNA revealed two different yeast EF-1 alpha genes and one or two different Drosophila genes, respectively . Nucleotide sequencing revealed signals for synthesis and processing of EF-1 alpha transcripts as well as the exact location of exons . One interruption in the coding sequence corresponds closely to a splice junction in the gene coding for the homologous chloroplast protein EF-Tu from Euglena gracilis, presumably of prokaryotic origin . The first exon in the chloroplast gene codes for the region of EF-Tu that is homologous to regions of the elongation factor EF-G and of the initiation factor IF2, respectively.

Science, 1986 Mar 7, 231(4742), 1154 - 7
An ancient developmental induction: heat-shock proteins induced in sporulation and oogenesis; Kurtz S et al.; Every eukaryotic and prokaryotic organism tested to date synthesizes a small number of heat-shock proteins in response to heat and other forms of stress . A particular pattern of heat-shock gene expression was observed during ascospore development in Saccharomyces: heat-shock proteins hsp26 and hsp84 were strongly induced nor inducible by heat shock . Instead, two proteins related to hsp70 were induced . A strikingly similar pattern of expression occurs during oogenesis in Drosophila, suggesting that it may be one of the earliest developmental pathways to evolve in eukaryotic cells.

Rev Infect Dis, 1986 Mar-Apr, 8(2), 208 - 17
Microbial ribosomal vaccines; Gregory RL; Ribosomal vaccines have been prepared from several different bacterial, fungal, and protozoan microorganisms . Most of these preparations offer a higher degree of protection than do vaccines made from the homologous whole cells, but the mode of protection is controversial . All of the reported ribosomal vaccines are contaminated with cell surface determinants . Antisera raised to ribosomal preparations are directed to innate ribosomal components as well as to surface antigens . Several hypotheses exist for the reported protection from disease: the cell surface contaminants serve as the protective moieties; ribosomes act as potent adjuvants for contaminating cell surface determinants; ribosomes innately contain antigenic determinants that cross-react with cell surface antigens; recently translated cell surface polypeptides are still attached to the ribosomal RNA in the ribosomes; ribosomes migrate from the cytoplasm of the microbe to the periphery of the cell, and ribosomal antigens are exposed on the cell surface; or ribosomes contain messenger RNA and produce microbial cell surface polypeptides in immunized individuals . A short description of the biochemical, biophysical, and structural characteristics of prokaryotic and eukaryotic ribosomes is presented, followed by a discussion of the ability of various ribosomal vaccines to protect against infectious agents.

Environ Health Perspect, 1986 Mar, 65, 71 - 5
Physiological and chemical characterization of cyanobacterial metallothioneins; Olafson RW; Techniques have been developed for detection, quantitation, and isolation of bacterial metallothioneins (MTs) from cyanobacterial species . These methods involve differential pulse polarography and reverse-phase high-performance liquid chromatography (HPLC) and have allowed detection of picomole quantities of these high sulfhydryl content proteins . The prokaryotic molecule was found to be induced in the presence of Cd or Zn salts with regulation at the level of transcription . Cu was not found to induce synthesis of the prokaryotic MT . Exposure to the former metals resulted in a growth lag followed by simultaneous induction of MT synthesis and onset of growth . Amino acid analysis and N-terminal sequence analysis indicated that the bacterial MTs from cyanobacteria are unique, having many aromatic and aliphatic residues and no apparent association of hydroxylated or basic amino acids with cysteines . Although the characteristic Cys-X-Cys sequences were present, no apparent amino acid sequence homology with the eukaryotic MTs was found in the first 42 residues.

Genetika, 1986 Mar, 22(3), 357 - 67
{Enhancer-like structures in moderately repetitive sequences of eukaryotic genomes}; Shakhmuradov IA et al.; The results of contextual analysis of 25 different middle repetitive DNA sequences are presented . It was shown that each of these repetitive DNA sequences contains at least one enhancer-like structure homologous to real enhancers, as well as to their consensus . The enhancer-like structures have been also revealed in the replication origin of some prokaryote genomes . The results are discussed in the light of a possible role of middle repetitive DNA sequences in the modulation of gene expression . Some aspects of genomes' evolution, in relation to enhancers, are also considered.

Mutat Res, 1986 Mar, 160(1), 1 - 10
The effect of gamma-irradiation on mu DNA transposition and gene expression; Kupelian A et al.; Mobile genetic elements are a ubiquitous presence in the genomes of all well-studied organisms . The effect of genomic stress on the status and transposition of these elements has not, as yet, been extensively characterized . We have been using temperate, transposable bacteriophage Mu as a model system to examine the behavior of mobile genetic elements and have previously shown that many DNA-damaging agents did not induce a Mu prophage to enter the lytic cycle of multiple rounds of DNA transposition . To extend these results and to examine the possibility that they were a reflection of damage to the DNA substrate for Mu transposition, we have constructed a mini-Mu plasmid, pMD12, which contains the early region of Mu, flanked by both extremities required for transposition in cis, and the beginning of the transposase gene A fused in frame to the lacZ gene . This A'-lacZ fusion protein maintains beta-galactosidase enzymatic activity under the control of the expression of the Mu transposase A gene and thus, the capacity for Mu transposition can be easily monitored by assaying for beta-galactosidase . By measuring the amount of beta-galactosidase after various doses of gamma-irradiation, we found that doses of up to 75 krad had no effect on the expression of the Mu transposase gene A . This was confirmed by the lack of induction of a Mu prophage in strains containing a chromosomally inserted Mu genome . Although the plaque-forming units per colony-forming unit of strain CSH67, containing a chromosomally inserted lambda prophage, increased approximately 100-fold from 0 to 75 krad, no stimulation of induction of prophage Mu lytic growth was observed . We also found that plasmid pMD12 did not transpose and chromosomally associate upon gamma-irradiation . This supports the assertion that DNA-damaging agents, including gamma-rays, do not induce the transposition of prokaryotic mobile genetic elements.

Mol Pharmacol, 1986 Mar, 29(3), 288 - 92
Effects of mercury (II) compounds on the activity of dUTPases from various sources; Williams MV; The deoxyuridine triphosphate nucleotidohydrolases (dUTPases, EC 3.6.1.23) from Escherichia coli K-12-,Acholeplasma laidlawii B-PG9-, human KB cell-, and the herpes simplex virus (HSV) type 1- and 2-induced dUTPases were purified and used to determine the effect of various mercury (II) compounds on their activities . Mercuric acetate, 5-mercuri-dUTP (HgdUTP), and 5-mercuri-dCTP (HgdCTP) acted as irreversible active site-directed inhibitors of the dUTPases purified from eukaryotic organisms but not those from prokaryotic organisms . The inhibition constants (Ki) were estimated for the KB, HSV-1, and HSV-2 dUTPases to be 8 +/- 2, 12 +/- 3, and 9 +/- 2 microM for mercuric acetate, 204 +/- 25, 121 +/- 15, and 111 +/- 10 microM for HgdUTP, and 775 +/- 25 and 651 +/- 23 microM for HgdCTP, respectively . The conversion of HgdUTP to its mercurithio-derivative resulted in a decrease in the affinity of the derivative for the eukaryotic dUTPases . The 5-mercurithioethylene glycol derivative of dUTP did not act as a substrate for the KB dUTPase but it did act as a substrate for the HSV-1- and HSV-2-induced dUTPases with Ki values of 526 +/- 47 and 483 +/- 32 microM, respectively . These results demonstrate that the eukaryotic dUTPases can be distinguished based upon differences in their affinities for the mercurithio-derivatives of dUTP and suggest that there are differences in the steric binding properties of the nucleotide-binding site of these enzymes.

Proc Natl Acad Sci U S A, 1986 Mar, 83(6), 1598 - 1602
Trans effect of the E1 region of adenoviruses on the expression of a prokaryotic gene in mammalian cells: resistance to 5' -CCGG- 3' methylation; Langner KD et al.; The plasmid construct pSVO-CAT has been used to test adenovirus promoter activities in the unmethylated or methylated state . We have now observed that the E2A late promoter of adenovirus type 2 (Ad2) DNA also activated the chloramphenicol acetyltransferase (CAT) gene upon transfection of the pAd2E2A-CAT construct into mammalian cells, and it was inactivated by specific methylations of three 5' -CCGG- 3' sites . Similar results had been reported previously after microinjecting promoter-methylated constructs into oocytes of Xenopus laevis . Surprisingly, it was found that the pSVO-CAT construct, which lacked eukaryotic promoter sequences, was able to express the CAT gene upon transfection into human or hamster cells that harbored and constitutively expressed the E1 region of Ad2 or Ad5 DNA . In these cells, the expression of the pAd2E2A-CAT construct was enhanced, but it was only partly sensitive to DNA methylation, possibly because DNA methylation was counteracted directly or indirectly by E1 functions . The pSVO-CAT construct was also expressed in HeLa or BHK21 cells upon cotransfection with a plasmid carrying the HindIII-G fragment of Ad2 DNA that contained the E1A region and part of the E1B region . By mapping pSVO-CAT-specific RNAs, we could demonstrate that pSVO-CAT activity in Ad2- or Ad5-transformed cells was mediated by prokaryotic promoter-like sequences in the pBR322 section of the construct, and it presumably functioned via trans-activation mediated by the E1 region . This trans-activation of pSVO-CAT in adenovirus-transformed cells was partly insensitive to DNA methylation.

Biochimie, 1986 Mar, 68(3), 459 - 70
Structure and function of cytochrome-c oxidase; Denis M; Recent works on the structure and the function of cytochrome-c oxidase are reviewed . The subunit composition of the mitochondrial enzyme depends on the species and is comprised of between 5 and 13 subunits . It is reduced to 1 to 3 subunits in prokaryotes . The complete amino acid composition has been derived from protein sequencing . Gene sequences are partially known in several eukaryote species . Metal centers are only located in subunits I and II . The mitochondrial cytochrome-c oxidase is Y-shaped; the arms of the Y cross the inner membrane, the stalk protrudes into the intermembrane space . The bacterial enzyme has a simpler, elongated shape . A number of data have been accumulated on the subunit topology and on their location within the protein . All available spectrometric techniques have been used to investigate the environment of the metal centers as well as their interactions . From the literature, attention must be paid to what may be considered or not as an active form . The steady improvement of the instrumentation has yielded evidence for different kinds of heterogeneities which could reflect the in vivo situation . The 'pulsed' and 'resting' conformers have been well characterized . The 'oxygenated' form has been identified as a peroxide derivative of the fully oxidized cytochrome-c oxidase . The mammalian enzyme has been isolated in fully active monomeric form which does not preclude the initially suggested dimeric behavior in situ . The role of the lipids is still largely investigated, mainly through reconstitution experiments . Kinetic studies of electron transfer between cytochrome c and cytochrome-c oxidase lead to a single catalytic site model to account for the multiphasic kinetics . Results related to the low temperature investigation of the intermediate steps in the reaction between oxygen and cytochrome-c oxidase received a sound confirmation by the resolution of compound A at room temperature . It is also pointed out that the so-called mixed valence state might not be a transient state in the catalytic reduction of oxygen . The functioning of cytochrome-c oxidase as a proton pump has been supported by a number of experimental results . Subunit III would be involved in this process . The redox link to the proton pump has been suggested to be at the Fea-CuA site . The molecular mechanism responsible for the proton pumping is still unknown.

J Mol Biol, 1986 Feb 20, 187(4), 581 - 90
Computer-averaged views of the 70 S monosome from Escherichia coli; Verschoor A et al.; The prokaryotic (70 S) monosome, composed of a roughly hemispherical 50 S large subunit and an elongate 30 S small subunit, appears in the electron micrograph in only a few common views representing the small number of preferred orientations assumed by the particle . Two of these, termed O and L views, have previously been characterized as the overlap and non-overlap projections; a third view, which we term the R view, represents the other endpoint of a rotational continuum with the overlap or O view . Tilt studies enabled us to calibrate this range as spanning approximately 50 degrees . The disjunct set of L views was averaged, and the reproducible resolution was determined to be 1/3.5 nm-1 . The combined sets of O and R views were analyzed by correspondence analysis, and a continuous "rotation series" of subaverages was obtained . Interpretation of the views in the light of what is known about the morphologies of the individual subunits allows a general picture of the mutual fit of the subunits in the monosome to be conceived.

Eur J Biochem, 1986 Feb 17, 155(1), 27 - 32
Proteins from the prokaryotic nucleoid . A protein-protein cross-linking study on the quaternary structure of Escherichia coli DNA-binding protein NS (HU); Losso MA et al.; Escherichia coli DNA-binding proteins NS1, NS2 and NS (NS1 + NS2) react with the protein-protein bifunctional cross-linking reagents dimethylsuberimidate and dimethyladipimidate to yield oligomers up to hexamers . The former reagent, with the longer arm, is more efficient than the other shorter one . Both one- and two-dimensional gel electrophoreses show that the cross-linked trimers are homogeneous, while the dimers appear heterogeneous, suggesting that at least two types of dimers but geometrically equivalent trimers are formed . In the presence of DNA, the cross-linking reaction with either reagent yields fewer dimers and more of the larger products . The yield of cross-linked products of various sizes was determined for NS1, NS2 and NS as a function of the protein concentration (0.03-3000 microM) . From the results obtained in these experiments, we derived a model of quaternary structure in which dimers and tetramers are predominant in very solutions of the proteins . Above a critical concentration (10-50 microM), interactions among tetramers become increasingly important, yielding octamers and perhaps larger products . Our data do not support a recently proposed model in which the DNA is packaged around a protein disc consisting of 8-10 NS dimers.

Cell, 1986 Feb 14, 44(3), 381 - 9
A bacteriophage RNA polymerase transcribes through a Xenopus 5S RNA gene transcription complex without disrupting it; Wolffe AP et al.; Conditions are described for programming with active transcription complexes essentially all the Xenopus 5S RNA genes added to an extract of Xenopus oocyte nuclei . We call this rate enhancement . Transcription by SP6 RNA polymerase of either strand of a Xenopus 5S RNA gene is unimpeded by the presence of a complete transcription complex associated with the internal control region of the gene . Furthermore, the transcription complex remains stably associated with the 5S RNA gene following multiple transits by the prokaryotic polymerase.

Biochem Cell Biol, 1986 Feb, 64(2), 154 - 60
Electrophoretic determination of sulfhydryl groups and its application to complex protein samples, in vitro protein synthesis mixtures, and cross-linked proteins; Stan-Lotter H et al.; Without prior fractionation, the number of sulfhydryl groups of individual polypeptides in a protein mixture can be determined, provided their molecular weights and approximate isoelectric points are known . Urea-denatured protein samples are reacted with iodoacetamide and iodoacetate in a modified version of Creighton's procedure . After separation by sodium dodecyl sulfate - polyacrylamide gel electrophoresis and isoelectric focusing, the number of sulfhydryl groups is determined by counting the protein bands which have additional negative charges . This method requires little material and provides an additional parameter, besides the molecular weight and isoelectric point, for the identification and characterization of a protein . The sensitivity may be enhanced for nonradioactive proteins by using 14C-labeled iodoacetamide and iodoacetate . The procedure has been applied to prokaryotic in vitro protein synthesis mixtures, bacterial membrane protein, and trypsin-cleaved or chemically cross-linked subunits of the F1 ATPase from Escherichia coli.

EMBO J, 1986 Feb, 5(2), 427 - 31
Requirements for substrate recognition by bacterial leader peptidase; Dierstein R et al.; Many secreted and membrane proteins have amino-terminal leader peptides which are essential for their insertion across the membrane bilayer . These precursor proteins, whether from prokaryotic or eukaryotic sources, can be processed to their mature forms in vitro by bacterial leader peptidase . While different leader peptides have shared features, they do not share a unique sequence at the cleavage site . To examine the requirements for substrate recognition by leader peptidase, we have truncated M13 procoat, a membrane protein precursor, from both the amino- and carboxy-terminal ends with specific proteases or chemical cleavage agents . The fragments isolated from these reactions were assayed as substrates for leader peptidase . A 16 amino acid residue peptide which spans the leader peptidase cleavage site is accurately cleaved . Neither the basic amino-terminal region nor most of the hydrophobic central region of the leader peptide are essential for accurate cleavage.

Exp Cell Res, 1986 Feb, 162(2), 285 - 95
Eukaryotic DNA metabolism . Are deoxyribonucleotides channeled to replication sites?
Mathews CK, Slabaugh MB.
DNA precursor biosynthesis is closely coordinated with DNA replication itself . In prokaryotic systems, firm evidence supports the idea that this coordination is achieved through the action of multienzyme complexes that physically link the synthesis of deoxyribonucleotides with their utilization in DNA replication . Much evidence favors a similar channeling mechanism in eukaryotes . However, recent studies suggest strongly that in mammalian cells DNA precursors are synthesized in cytoplasm and are then transported into the nucleus . This article reviews the pertinent evidence, attempts to reconcile contradictory findings, and highlights areas that need further investigation.

J Biomol Struct Dyn, 1986 Feb, 3(4), 725 - 38
Contextual constraints on codon pair usage: structural and biological implications; Kolaskar AS et al.; Complementary DNA sequence data of 278 protein coding genes from prokaryotic systems have been analysed at the level of near neighbour codon pairs . Our analysis points out that constraints exist even at the level of near neighbour codon pairs . These constraints are in addition to those which arise due to relative levels of tRNA . Codon pairs, which in the data base have different occurrence values from their expected values, neither have common secondary structure nor do have better stabilization due to high base stacking . Our study points out that there are strong interaction between constituent codons in these codon pairs . These strongly interacting codon pairs, we suggest, are involved in the formation of three dimensional structural elements of cDNA/mRNA and interact with ribosome and thus modulate translation.

J Biomol Struct Dyn, 1986 Feb, 3(4), 705 - 23
Terminators of transcription with RNA polymerase from Escherichia coli: what they look like and how to find them; Brendel V et al.; We present here a compilation of prokaryotic transcription terminator sequences (ref . 1-152) . The compilation includes 49 independent terminators, 52 speculated independent terminators, 27 sites shown to function in vivo, and some 20 proven or speculated rho-dependent terminators . In addition to the well-known features of independent terminators (dyad symmetry and T-run), two consensus are found: CGGG(C/G) upstream and TCTG downstream of the termination point . A subset of the collection of sequence has been used to construct a computer algorithm to locate independent terminators by sequence analysis.

Cell, 1986 Jan 31, 44(2), 243 - 9
Reconstitution of RNAase P activity using inactive subunits from E . coli and HeLa cells; Gold HA et al.; HeLa cell RNAase P activity found in the flow-through of anti-Sm affinity columns can be separated into inactive RNA and protein components . These components can be used to reconstitute active hybrid enzyme complexes with purified subunits from E . coli RNAase P . The RNA in the HeLa cell fractions employed is enriched for species between 85 and 115 nucleotides long . This reconstitution assay is a convenient means of purifying the functional RNA and protein of HeLa cell RNAase P . Probes derived from the genes for the subunits of E . coli RNAase P hybridize to genomic DNA of gram-negative prokaryotic organisms, but no positive signals are seen with genomic DNA from a variety of eukaryotic organisms.

J Biol Chem, 1986 Jan 15, 261(2), 982 - 7
cDNA and deduced protein sequence for the beta B1-crystallin polypeptide of the chicken lens . Conservation of the PAPA sequence; Hejtmancik JF et al.; The nucleotide sequence of two cDNA clones corresponding to the beta B1-crystallin mRNA (formerly beta 35) of the chicken eye lens has been determined . The derived amino acid sequence of the chicken beta B1 polypeptide fits well with the two-domain, four "Greek Key" motif structure common to the beta gamma-crystallin superfamily of proteins . The calculated molecular weight of the encoded chicken beta B1 protein is 27,267 . The beta B1 polypeptide has both an N- and C-terminal extension and is highly homologous to the mammalian beta B1-crystallin polypeptide . There is a 72% homology between the core regions of the chicken and bovine beta B1 polypeptides; by contrast, there is only 27% homology between the N-terminal extensions of these polypeptides . The N-terminal extension of chicken beta B1 contains a short alternating proline-alanine (PAPA) sequence, like that in the mammalian beta B1, and has some homology with the N-terminal region of histone H1.4, myosin light chain, prokaryotic outer membrane protein A, and adenovirus 24/28-kDa early protein . At the nucleic acid level, the chicken beta B1 crystallin gene has an atypical polyadenylation signal, AATTAAA . This appears to be associated with microheterogeneity of the polyadenylation site by comparison of two cDNA clones, suggesting an additional level at which diversity in crystallin gene expression may arise.

Eur J Biochem, 1986 Jan 15, 154(2), 355 - 62
Structural homology between elongation factors EF-Tu from Bacillus stearothermophilus and Escherichia coli in the binding site for aminoacyl-tRNA; Jonak J et al.; Elongation factor EF-Tu (Mr approximately equal to 50 000) and elongation factor EF-G (Mr approximately equal to 78 000) were isolated from Bacillus stearothermophilus in a homogeneous form . The ability of EF-Tu to participate in protein synthesis is rapidly inactivated by N-tosyl-L-phenyl-alanylchloromethane (Tos-PheCH2Cl) . EF-Tu X GTP is more susceptible to the inhibition by Tos-PheCH2Cl than is EF-Tu X GDP . Tos-PheCH2Cl forms a covalent equimolar complex with the factor by reacting with a cysteine residue in its molecule . The labelling of EF-Tu by the reagent irreversibly destroys its ability to bind aminoacyl-tRNA, which in turn protects the protein from this inactivation . This indicates that the modification of EF-Tu by Tos-PheCH2Cl occurs at the aminoacyl-tRNA binding site of the protein . To identify and characterize the site of aminoacyl-tRNA binding in EF-Tu, the factor was labelled with {14C}Tos-PheCH2Cl, digested with trypsin, the resulting peptides were separated by high-performance liquid chromatography and the sequence of the radioactive peptide was determined . The peptide has identical structure with an Escherichia coli EF-Tu tryptic peptide comprising the residues 75-89 and the Tos-PheCH2Cl-reactive cysteine at position 81 {Jonak, J., Petersen, T . E., Clark, B . F . C . and Rychlik, I . (1982) FEBS Lett . 150, 485-488} . Experiments on photo-oxidation of EF-Tu by visible light in the presence of rose bengal dye showed that there are apparently two histidine residues in elongation factor Tu from B . stearothermophilus which are essential for the interaction with aminoacyl-tRNA . This is clearly reminiscent of a similar situation in E . coli EF-Tu {Jonak, J., Petersen, T . E., Meloun, B . and Rychlik, I . (1984) Eur . J . Biochem . 144, 295-303} . Our results provide further evidence for the conserved nature of the site of aminoacyl-tRNA binding in elongation factor EF-Tu and show that Tos-PheCH2Cl reagent might be a favourable tool for the identification of the site in the structure of prokaryotic EF-Tus.

J Biol Chem, 1986 Jan 15, 261(2), 874 - 7
A human gene family with sequence homology to Drosophila melanogaster Hsp70 heat shock genes; Mues GI et al.; A growing literature describes the structure and regulation of prokaryotic and eukaryotic heat shock genes . We here report the isolation of several members of a human heat shock protein 70 (hsp 70) multigene family which contains at least 10 different genes and/or pseudogenes exhibiting sequence homology to the hsp70 gene of Drosophila melanogaster . Eight nonoverlapping recombinant lambda phages from a lambda-Charon4A human genomic library were studied by restriction mapping . One lambda clone was sequenced and characterized as a hsp70 pseudogene inserted into a rearranged human HindIII 1.9-kilobase repeated DNA sequence . This pseudogene is probably located on the X chromosome . Its predicted amino acid sequence shows extensive homology to those of Drosophila hsp70, trout hsp70, Xenopus hsp70, yeast hsp70, and some homology to the heat-inducible dnaK gene product of Escherichia coli . Amino acid homology is clustered, suggesting evolutionary conservation of domains critical to the function of this protein in both prokaryotic and eukaryotic cells.

J Mol Biol, 1986 Jan 5, 187(1), 47 - 60
Interaction of the Escherichia coli HU protein with DNA . Evidence for formation of nucleosome-like structures with altered DNA helical pitch; Broyles SS et al.; Nuclease digestion studies of DNA bound to the histone-like protein HU show that cuts in each strand of the DNA double helix are made with a periodicity of 8.5 base-pairs . By contrast, similar digestions of DNA in eukaryotic nucleosomes show a repeat of 10.4 base-pairs . This and other results (including circular dichroism studies) are consistent with the proposal that the pitch of the DNA double helix in the HU complex is reduced from a repeat length of 10.5 to 8.5 base-pairs per helical turn . Simultaneously, the DNA in the HU-DNA complex containing two dimers of HU per 60 base-pairs has its linking number decreased by 1.0 turn per 290 base-pairs . From these changes it is calculated that HU imposes a DNA writhe of 1.0 per three to four monomers of HU . The results suggest a model in which DNA is coiled in left-handed toroidal supercoils on the HU complex, having a stoichiometry resembling that of the half-nucleosome of eukaryotic chromatin . An important distinction is that HU complexes can restrain the same number of DNA superhelical turns as eukaryotic nucleosomes, yet the DNA retains more negative torsional tension, just as is observed in prokaryotic chromosomes in vivo . Another distinction is that HU-DNA complexes are less stable, having a dissociation half-life of 0.6 min in 50 mM-NaCl . This last property may explain prior difficulties in detecting prokaryotic nucleosome-like structures.

Eur J Biochem, 1986 Jan 2, 154(1), 193 - 6
Sequence determinants of cytosolic N-terminal protein processing; Flinta C et al.; N-terminal methionine removal has been analyzed statistically in a large sample of prokaryotic and eukaryotic cytosolic proteins in an attempt to uncover common sequence determinants . We find that the residue next to the initiator Met is the most important determinant of N-terminal processing: Lys, Arg, Leu and (in prokaryotes) Phe and Ile protect the initiator Met from being removed when next to it in the sequence; Ala, Gly, Pro, Ser, Thr and (in eukaryotes) Val in this position cause its removal . Subsequent acetylation is confirmed to be strongly biased towards Ala, Met and Ser residues; when Met is acetylated, Asp is the predominant penultimate residue in eukaryotes . Also, we find major differences in the relative abundance of the various residues next to the initiator Met between prokaryotes and eukaryotes: prokaryotic proteins are much more biased towards Lys as the Met-protecting residue, and towards Ala when met is to be removed, than eukaryotic ones . Finally, we show that our results can explain a part of the mRNA 'consensus sequence' found around eukaryotic initiator AUG codons.

Comp Immunol Microbiol Infect Dis, 1986, 9(2-3), 131 - 6
Synthetic immunostimulants (excluding sulfur-containing compounds); Werner GH; Many different approaches have been used, over the last 15 years, for the design of potential immunostimulating drugs: Fractionation of crude natural substances (of eukaryotic or prokaryotic origin) already known to enhance immune functions, followed by chemical characterization and, in many cases, full synthesis of the active moiety: examples are provided by thymic hormones and the muramyldipeptide (MDP); Chemical modification of natural substances of known chemical structure in order to potentiate or change their biological activities or reduce their toxicity: murabutide, lipophilic MDP derivatives, lipopeptides (such as pimelautide), tuftsin analogs; Chemical synthesis (often without preconceived ideas about structure-activity relationship) of a great variety of molecules which are then screened in vitro and in vivo for immunopharmacological activity . The chemical structures and the biological profiles (in terms of possible primary cellular targets and mechanisms of immunostimulating activities) of representatives of class (b) and class (c) immunostimulants are reviewed in this paper.

Microbios, 1986, 48(196-197), 173 - 82
Immunological detection of hCG-like substances in aerobic bacteria of both tumour and non-tumour origin; Affronti LF et al.; Production of hormone-like substances by prokaryotes and unicellular eukaryotes, as well as the presence of a human chorionic gonadotropin-like substance in the serum of individuals with malignancies, have been previously established . To determine the presence and distribution of the production of human chorionic gonadotropin (hCG) among aerobic bacteria, fourteen strains of bacteria of both tumour and non-tumour origin were examined using enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and indirect immunoperoxidase techniques . Of the six bacterial strains tested, half of the tumour-isolates produced an hCG-like substance while non-tumour associated strains of the same species did not do so . Virulent strains of two mycobacterial species also gave high positive values while avirulent Mycobacterium tuberculosis strains (H37Ra) yielded low or negative values . The production of hCG-like substances by mycobacteria, for which no tumour association is known, indicates that tumour association is neither a prerequisite nor a requirement for production of hCG . It appears that the presence of hCG-like substances is a variable character among bacterial species, suggesting that it may have been conserved throughout evolutionary history.

Annu Rev Microbiol, 1986, 40, 55 - 77
Tryptophan biosynthetic genes in eukaryotic microorganisms; Hutter R et al.; In recent years more information about tryptophan biosynthesis in eukaryotic microorganisms has become available . The emphasis has been on genetics and biochemistry of the pathway . Eukaryotes manifest a trend toward fewer genes and toward multifunctional proteins, while prokaryotes have a greater tendency toward separate activity domains but the genes tend to be clustered genetically . Cloning of various structural tryptophan biosynthetic genes and studies on their expression in homologous and heterologous hosts have made it possible to analyze promoter structures in detail and to define structural elements involved in regulated gene expression . Comparisons of homologous genes from different organisms have highlighted the conservation of the activity domains or parts therefrom involved in the catalysis of single steps . These studies also point to a stringent maintenance of domains responsible for protein-protein aggregation . Physiological studies will be facilitated by the availability of single cloned genes and especially the artificial gene cluster containing all five TRP genes from yeast . The range of physiological manipulation has thus been enormously broadened . With chromosomal mutations it has been possible to study primarily downward modulation of a pathway . We can now initiate studies on upward modulation, since enzyme levels appear to increase in proportion to gene dose . The new range of downward and upward modulation in the levels of single enzymes and combinations of enzymes may contribute to a better understanding of flux regulation and its influence on the overall physiology of an organism.

J Cell Biochem, 1986, 32(1), 35 - 49
The versatility of proteolytic enzymes; Neurath H; The growing realization of their physiological importance has generated renewed interest in the study of proteolytic enzymes . Modern methods of protein chemistry and molecular biology have revealed new insights into the protein and gene structure of a variety of protein precursors and their processing by limited proteolysis . Examples are given in this review for transmembrane processes and the role of signal peptidases of both eukaryotic and prokaryotic origin, the processing of prohormones and precursors of growth factors, protein components of blood coagulation, fibrinolysis, and of the complement system, and a group of granulocyte proteases, including the mast cell serine proteases . The relationship of homologous domains found in many of these proteases and their zymogens to protein evolution is a recurrent theme of this discussion.

Eur Biophys J, 1986, 13(5), 301 - 7
Comparison of the structure of ribosomal 5S RNA from E . coli and from rat liver using X-ray scattering and dynamic light scattering; Muller JJ et al.; The structure of eukaryotic ribosomal 5S RNA from rat liver and of prokaryotic 5S RNA from E . coli (A-conformer) have been investigated by scattering methods . For both molecules, a molar mass of 44,500 +/- 4,000 was determined from small angle X-ray scattering as well as from dynamic light scattering . The shape parameters of the two rRNAs, volume Vc, surface Oc, radius of gyration Rs, maximum dimension of the molecule L, thickness D, and cross section radius of gyration Rsq, agree within the experimental error limits . The mean values are Vc = 57 +/- 3 nm3, Oc = 165 +/- 10 nm2, Rs = 3.37 +/- 0.05 nm, L = 10.8 +/- 0.7 nm, D = 1.57 +/- 0.07 nm, Rsq = 0.92 +/- 0.01 nm . Identical structures for the E . coli 5S rRNA and the rat liver 5S rRNA at a resolution of 1 nm can be deduced from this agreement and from the comparison of experimental X-ray scattering curves and of experimental electron distance distribution functions . The flat shape model derived for prokaryotic and eukaryotic 5S rRNA shows a compact region and two protruding arms . Double helical stems are eleven-fold helices with a mean base pair distance of 0.28 nm . Combining the shape information obtained from X-ray scattering with the information about the frictional behaviour of the molecules, deduced from the diffusion coefficients D020, w = (5.9 +/- 0.2) X 10(-7) cm2 s-1 and (6.2 +/- 0.2) X 10(-7) cm2 s-1 for rat liver 5S rRNA and E . coli 5S rRNA, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Annu Rev Nutr, 1986, 6, 155 - 78
Gastrointestinal microflora in mammalian nutrition; Savage DC; A mammal is a complex organism consisting of eukaryotic animal cells and eukaryotic and prokaryotic microbial cells . Most of the microorganisms reside in communities in the gastrointestinal tract . These gastrointestinal microfloras are known to serve nutritional functions in ruminants, pseudoruminants, and monogastric mammals with only modest or no foregut fermentations but with extensive hindgut fermentations in blind cecal pouches . In adult animals, the microflora hydrolyzes exogenous (dietary) and endogenous polymers, and provides the adult with all or at least a significant proportion of its carbon, energy, vitamins, and macromolecular building blocks . The flora also functions as a conservator of nitrogen that would otherwise be excreted as urea . In exchange, the flora competes directly with the host tissues for nutrients ingested in the diet, and also competes indirectly by somewhat repressing the absorptive capacities of the animal tissues . When the synergism is in balance, the animal tissues and the microflora operate in harmony for the health and nutritional welfare of the host as a whole . The system may be unbalanced by antibacterial drugs that destroy the microflora and by diseases of the animal tissues that destroy the controls regulating where indigenous communities localize in the tract, their microbial composition, and their biochemical activities . At such times, the nutrition of the animal tissues can be adversely affected to the extreme . Humans living in developed and developing countries have extensive microfloras in their hindguts . Humans living in developing countries may also have extensive microfloras in their small bowels . Those floras may function in nutrition of the animal tissues of man much the same as do floras in similar locations in the gastrointestinal tracts of mammals other than man . However, animals of some species other than human gain much of the nutritional benefit from their microflora through the practice of coprophagy . Since adult humans do not normally practice coprophagy, any nutritional benefit from the microflora depends upon the capacity of the bowel mucosa, principally that of the large bowel, to absorb bacterial products, e.g . short-chain volatile fatty acids . Such absorption undoubtedly occurs, but is surely not a major source of carbon and energy for the animal tissues of man.

Drugs Exp Clin Res, 1986, 12(4), 335 - 42
Bactericidal effects induced by laser irradiation and haematoporphyrin against gram-positive and gram-negative microorganisms; Martinetto P et al.; Laser irradiation of tissues treated in vivo with haematoporphyrin (Hpr) is known to result in a cytocidal effect, reportedly more pronounced in tumour tissues . To ascertain whether this cytocidal phenomenon can occur not only in eukaryotic but also in prokaryotic cells, the authors devised a model system in vitro consisting of bacterial cultures in liquid and in solid media . Bacteria photosensitized with Hpr were subsequently exposed to laser beams and to daylight; then normal microbiological techniques were used to determine whether any bactericidal effect occurred . Satisfactory results were achieved, particularly against Gram-positive microorganisms; a partial inhibition of Gram-negative microorganisms was also observed.

Proc Natl Acad Sci U S A, 1986 Jan, 83(1), 24 - 7
Hemoglobin Long Island is caused by a single mutation (adenine to cytosine) resulting in a failure to cleave amino-terminal methionine; Prchal JT et al.; Hemoglobin Long Island has two separate amino acid abnormalities of beta-globin structure: an extension of the NH2 terminus by a methionine residue and a histidine-to-proline substitution at the normal second position . The NH2-terminal methionine residue, the translation product of an AUG initiation codon, is present only transiently in nascent proteins . Because of the general biological implications of this abnormality, we investigated the nature of the genetic defect of this mutant . We determined the sequence of the relevant portion of the beta-globin mRNA by means of dideoxynucleotide chain termination of the complementary DNA (cDNA) in which an oligonucleotide complementary to codons 10-17 was used as a primer for reverse transcriptase . A histidine-to-proline substitution was confirmed in the mutant mRNA by identifying an adenine-to-cytosine transversion in the second codon . However, we were unable to find any other abnormality at either the AUG initiation codon or in the 56 bases upstream from the adenine-to-cytosine transversion (encompassing most of the 5' untranslated region of the mutant beta-globin mRNA) . Thus, it appears that this single lesion probably interferes with the poorly understood methionine-cleaving mechanism that modulates most of prokaryotic and eukaryotic proteins.

Curr Genet, 1986, 10(11), 819 - 22
Prokaryotic promoters in the chloroplast DNA replication origin of Chlamydomonas reinhardtii; Wu M et al.; In Chlamydomonas reinhardtii, one displacement loop region which initiates the replication of chloroplast DNA was located on a 1.05 kb restriction fragment . This fragment was cloned and sequenced . In this report, the galK expression plasmid, pKO1 was used to screen for the presence of any prokaryotic promoter within the cloned fragment . The insertion of 2 AluI fragments yielded galK+ colonies . Sequence analyses of these AluI inserts revealed prokaryotic promoter consensus regions . Cloning into pKOTWI and subsequent DNA sequencing were used to determine the promoter-active orientation of each insert . Two back-to-back prokaryotic promoters were mapped on a 79 bp AluI fragment located within the displacement loop region.

Curr Genet, 1986, 10(5), 343 - 52
Yeast adenylate cyclase catalytic domain is carboxy terminal; Masson P et al.; Subcloning of DNA fragments from the gene coding for yeast adenylate cyclase has permitted, after complementation studies in S . cerevisiae cdc35 mutants as well as E . coli cya mutants, to identify the sequence coding for the catalytic domain of the protein . No homology is found between the yeast cyclase catalytic domain and the homologous domain found in E . coli adenylate cyclase . Analysis by Northern blotting of yeast polyA mRNA has shown the existence of multiple transcriptional products of the gene . A putative origin of a major transcript (3.5 kb) would allow synthesis of a ca . 100,000 dalton protein exhibiting cyclase activity in its carboxy terminal domain, and having 7 repeats of 17 amino acids at its amino terminal end . Several note-worthy features, including the possibility of transcriptional control by the general control of amino acids biosynthesis, are present at this putative origin . Data are presented suggesting that a much longer gene product might also be synthesized from the CDC35 gene . Neither the gene organization nor the amino acid sequence of the protein does display any homology with the adenylate cyclase gene and protein of Escherichia coli . This suggests a case of evolutionary convergence for the synthesis of cAMP in prokaryotes and eukaryotes.

J Mol Evol, 1986, 24(1-2), 130 - 42
Protein export in prokaryotes and eukaryotes: indications of a difference in the mechanism of exportation; Gascuel O et al.; Investigation of possible variations between prokaryotic and eukaryotic signal sequences of exported proteins has revealed unexpected differences . Apart from the known similarities (presence of a core hydrophobic sequence preceded by a positively charged amino terminus and followed by a flexible structure), we have found that the core is much more rigid in eukaryotic signals than in their prokaryotic counterparts, and that at both ends the constraints are much more stringent in bacteria than in human cells . The differences have been summarized as a set of 17 criteria describing noteworthy features discriminating between the two classes of signal peptides . The program we used permitted each class of sequences to be learned; Escherichia coli sequences were well learned (i.e., they could be recognized by the programs as having common features), whereas human sequences were found to exhibit a much wider variation . Thus it was possible to propose a consensus in the case of the bacterial peptides, but none (or a much looser one) in the case of the human sequences . Two sequences were exceptional among the E . coli signal peptides, those of lipoprotein and plasmid-borne beta-lactamase, suggesting that they have special origins or destinations . Finally, the differences found strongly suggest that the mode of secretion is rather different in the two types of organisms, in spite of the common features of the signal sequences.

J Mol Evol, 1986, 23(4), 328 - 35
The complete amino acid sequences of cytosolic and mitochondrial aspartate aminotransferases from horse heart, and inferences on evolution of the isoenzymes; Doonan S et al.; We report here the complete amino acid sequences of the cytosolic and mitochondrial aspartate aminotransferases from horse heart . The two sequences can be aligned so that 48.1% of the amino acid residues are identical . The sequences have been compared with those of the cytosolic isoenzymes from pig and chicken, the mitochondrial isoenzymes from pig, chicken, rat, and human, and the enzyme from Escherichia coli . The results suggest that the mammalian cytosolic and mitochondrial isoenzymes have evolved at equal and constant rates whereas the isoenzymes from chicken may have evolved somewhat more slowly . Based on the rate of evolution of the mammalian isoenzymes, the gene-duplication event that gave rise to cytosolic and mitochondrial aspartate aminotransferases is estimated to have occurred at least 10(9) years ago . The cytosolic and mitochondrial isoenzymes are equally related to the enzyme from E . coli; the prokaryotic and eukaryotic enzymes diverged from one another at least 1.3 X 10(9) years ago.

J Mol Evol, 1986, 23(4), 300 - 4
Nucleotide sequences of Cyanophora paradoxa cellular and cyanelle-associated 5S ribosomal RNAs: the cyanelle as a potential intermediate in plastid evolution; Maxwell ES et al.; The 5S ribosomal RNAs from the cell cytoplasm and cyanelle (photosynthetic organelle) of Cyanophora paradoxa have been isolated and sequenced . The cellular and cyanelle 5S rRNAs were 119 and 118 nucleotides in length, respectively . Both RNAs exhibited typical 5S secondary structure, but the primary sequence of the cellular species was clearly eukaryotic in nature, while that of the organellar species was prokaryotelike . The primary sequence of the cyanellar 5S rRNA was most homologous to cyanobacterial 5S sequences, yet possessed secondary-structural features characteristic of higher-plant chloroplast 5S rRNAs . Both sequence comparison and structural analysis indicated an evolutionary position for cyanelle 5S rRNA intermediate between blue-green alga and chloroplast 5S rRNAs.

Basic Life Sci, 1986, 39, 231 - 42
Nucleotide excision repair genes from the yeast Saccharomyces cerevisiae; Friedberg EC et al.; The genetics of nucleotide excision repair in the yeast Saccharomyces cerevisiae is complex, apparently requiring at least 10 genes . We have isolated 5 of these genes (designated RAD1, RAD2, RAD3, RAD4, and RAD10) by molecular cloning and plan to overexpress them in order to generate proteins for biochemical study . We have sequenced four of these five genes and have noted regions of homology with other proteins in the predicted amino acid sequence of some of them . In particular, there is striking homology between Rad3 protein and a number of prokaryotic and eukaryotic proteins that bind nucleotides and hydrolyze ATP or GTP . Mutations in this region of the RAD3 gene render cells defective in the nucleotide excision repair function . In addition to its role in nucleotide excision repair, the RAD3 gene is essential for the viability of haploid cells in the absence of DNA damage . The nature of the essential function is unknown . The RAD1 and RAD3 genes are not inducible by DNA damaging agents . However, exposure of cells to UV radiation, 4-nitroquinoline 1-oxide, or gamma radiation results in 4- to 6-fold enhanced expression of the RAD2 gene.

Zentralbl Mikrobiol, 1986, 141(5), 415 - 9
Effect of root extract from Boerhaavia diffusa L., containing an antiviral principle upon plaque formation of RNA bacteriophages; Awasthi LP et al.; An extract obtained from the roots of Boerhaavia diffusa plants, which inhibits the infection of several plant viruses, was tested by the agar diffusion hole method for its action on RNA-containing bacterial viruses . Plaque formation of the phages was only partially and non-uniformly influenced by the extract so that a uniform principle of action was not realized for the RNA viruses of prokaryotic and eukaryotic host organisms.

Acta Biotheor, 1986, 35(1-2), 69 - 76
Evolution of cell and chromosome structure in eukaryote; Sharma AK; The analysis of the data so far available indicates that eukaryotic chromosome with splicing characteristics appeared quite early in evolution possibly parallel and not sequential to the prokaryotic system . The endosymbiotic origin of the eukaryotic cell involved a primitive undifferentiated unicellular eukaryote and a photosynthetic or non-photosynthetic microbe . Certain regulatory genes of extra-cellular organelles were transferred later through molecular hybridization to the nucleus . The evolution of multicellularity and sexual reproduction led to the origin of innumerable eukaryotic forms in the late precambrian period . This new concept of the author can account for the evolution of complex eukaryotic chromosome and harmonious functioning of extra-cellular organelles with the nucleus . The concept also explains the sudden spurt of innumerable eukaryotic fossils at the early palaeozoic era.

Gene, 1986, 49(1), 93 - 102
Chromosome analysis by two-dimensional fingerprinting; Poddar SK et al.; A two-dimensional fingerprinting technique has been developed that allows large, cell genome-size DNA's to be analyzed by restriction endonuclease cleavage and separation of DNA fragments by agarose gel electrophoresis . Equations have been derived to determine the genome size and number of cleavage sites from analysis of the distribution of fragment lengths . Genome sizes of Escherichia coli strain JM101 and two strains of the mycoplasma Acholeplasma laidlawii measured by this method are in agreement with published values . Other uses of two-dimensional fingerprinting for studies of prokaryotic and eukaryotic genome structure and organization are described.

Gene, 1986, 49(1), 69 - 80
Complete nucleotide sequence of the penicillin acylase gene from Kluyvera citrophila; Barbero JL et al.; The penicillin acylase (PAC) from Kluyvera citrophila ATCC21285 has been purified to homogeneity and found to be composed of two non-identical subunits of 23 and 62 kDa, in contrast with the previous findings {Shimizu et al., Agr . Biol . Chem . 39 (1975) 1655-1661} . The nucleotide (nt) sequence of the K . citrophila pac gene contained in the 3-kb PvuI-HindIII fragment of pKAP1 {Garcia and Buesa, J . Biotechnol . 3 (1986) 187-195} has been determined, showing that it encodes a protein of 844 amino acid (aa) residues . The aa analysis of the N-terminal and C-terminal sequences of the purified subunits showed that they were derived from a common precursor protein of 93.5 kDa, from which a signal peptide of 26 aa, responsible for the periplasmic translocation of the protein, and an internal connecting polypeptide of 54 aa, have been removed in the maturation of the PAC . The comparison of the nt sequences of the pac genes from K . citrophila and Escherichia coli ATCC11105 {Schumacher et al., Nucl . Acids Res . 14 (1986) 5713-5727} revealed 80% homology, suggesting a common ancestral pac gene origin . The results reported here should allow investigation of the unusual mechanism of maturation of this prokaryotic protein, as well as manipulation, using DNA recombinant techniques, of the catalytic properties of this industrially important enzyme.

Gene, 1986, 49(1), 153 - 6
Terminal inverted repeats of prokaryotic transposable element IS186 which can generate duplications of variable length at an identical target sequence; Sengstag C et al.; The insertion element IS186, which resides in the chromosome of Escherichia coli K-12, is 1338 bp long . Its termini represent 23-bp perfectly inverted repeats, but a variant carries a mismatch at position 23 . IS186 transposes preferentially into G + C-rich sequences and generates target duplications of variable length, even at the same integration site.

Biosystems, 1986, 19(4), 247 - 58
The evolution of eukaryotic ribosomal DNA; Gerbi SA; Mutations occur randomly throughout the ribosomal DNA (rDNA) sequence . Molecular drive (unequal crossing-over, gene conversion, and transposition) spreads these variations through the multiple copies of rDNA . Forces of selection act upon the variants to favor and fix them or disfavor and eliminate them . Selection has not permitted changes in regions within rRNA vital for its function; these sequences are evolutionarily conserved between diverse species . Possible functions for some of these conserved sequences are discussed . The secondary structure of rRNA is also highly conserved during evolution . However, eukaryotic rRNA is larger than prokaryotic rRNA due to blocks of "expansion segments" . Arguments are put forward that expansion segments might not play any functional role . Other examples are reviewed of rDNA sequence insertion or deletion, including introns and the internal transcribed spacer 2.

J Cyclic Nucleotide Protein Phosphor Res, 1986, 11(4), 253 - 64
Identification of a rat liver cAMP-dependent protein kinase, type II, which binds DNA; Shabb JB et al.; A novel protein kinase which specifically binds single strand DNA was identified in rat liver by chromatography on double strand- and single strand- DNA cellulose . This protein kinase activity was stimulated by cAMP and was inhibited by the heat stable inhibitor, suggesting it was a cAMP-dependent protein kinase . Isoelectric focusing studies confirmed that the single strand DNA-binding protein kinase was indeed a cAMP-dependent protein kinase and had the same pI as cAMP-dependent protein kinase, Type II . The DNA binding capacity of this kinase was primarily localized in the regulatory subunit . These results support the recent hypothesis that in addition to regulating enzymatic activity by phosphorylating proteins, cAMP-dependent protein kinase, Type II, may regulate mammalian gene expression through a mechanism similar to that in prokaryotes.

Gene, 1986, 43(1-2), 69 - 77
The fos gene product undergoes extensive post-translational modification in eukaryotic but not in prokaryotic cells; Sambucetti LC et al.; To investigate the properties of the fos oncogene, we have constructed bacterial and yeast vectors which express the entire fos-coded protein (Fos) and two C-terminal deletion products . In Escherichia coli, Fos proteins were expressed from the phage lambda pL promotor under the control of the temperature-sensitive lambda repressor . In vitro transcription/translation studies indicate that these vectors produce Fos proteins of the expected sizes . However, in vivo, Fos protein accumulation is observed only in hosts with the Lon- phenotype . In Saccharomyces cerevisiae, the fos gene was expressed from the PHO5 promoter which is induced under low-phosphate conditions . In contrast to the situation in E . coli, in which the heterologous proteins appeared as single major products when subjected to sodium dodecyl sulfate - polyacrylamide gel electrophoresis, the Fos proteins in S . cerevisiae displayed extensive Mr heterogeneity . Pulse-chase analyses indicated that this heterogeneity was a consequence of extensive post-translational modification . These modifications occurred to an equivalent extent on the products coded by the fos gene with C-terminal deletions and thus appear not to be controlled by the missing domain.

Carcinogenesis, 1986 Jan, 7(1), 185 - 8
Expression of the E . coli O6-methylguanine-methylphosphotriester methyltransferase gene in mammalian cells; Brennand J et al.; Many prokaryotic and eukaryotic cells contain enzymes that repair damage introduced into their DNA following exposure to chemical, physical and biological agents . One such lesion that has received considerable attention is the potentially miscoding and mutagenic base O6-alkylguanine which is produced in varying amounts in DNA following reaction with monofunctional alkylating agents . As part of a study to assess the role of this lesion and its repair in the processes of cytotoxicity, mutagenicity and transformation, we have recently cloned the Escherichia coli gene which codes for the protein responsible for the repair of such damage in DNA . In the present study we describe the construction of a plasmid which allows the efficient expression of the bacterial gene in mammalian cells.

Proc Natl Acad Sci U S A, 1986 Jan, 83(2), 217 - 20
Evidence against use of bacterial amino acid sequence data for construction of all-inclusive phylogenetic trees; Meyer TE et al.; It has been proposed that phylogenetic trees, intended to show divergence of eukaryotic protein and nucleic acid sequences, be extended to include those from bacteria . However, we have compared the amino acid sequences of 18 of the most divergent mitochondrial cytochromes c with those of 18 bacterial cytochromes c2 and have found that the average percentage difference between these mitochondrial cytochromes c and cytochromes c2 was not significantly greater than that among the cytochromes c2 alone . The large discontinuities in physical-chemical properties recognized between the prokaryote and eukaryote cytochromes render it highly improbable that members of the two classes should be no more different from one another than members of either class alone, assuming that sequence differences can accurately reveal evolutionary divergence . Instead, we propose that divergent amino acid sequences approach a limit of change considerably less than for comparison of random sequences . This limit of change presumably is determined by the structure/function relationship . When two homologous protein sequences have reached such a limit, convergence or back-mutations and parallel mutations become as frequent as divergent mutations . As two diverging proteins approach this steady-state condition, sequence differences no longer reflect the numbers of mutations resulting in amino acid substitution and therefore species cannot be positioned on a phylogenetic tree . Insertions and deletions are less reversible than are amino acid substitutions and, provided they are well-documented, might be more reliable indicators of bacterial relationships . Nevertheless, we suggest that data available on bacterial protein sequences do not permit construction of all-inclusive phylogenetic trees . Comparisons of protein and rRNA trees suggest that similar restrictions apply to use of rRNA sequence data.

Gene, 1986, 47(2-3), 269 - 77
Cyclodextrin-glycosyltransferase from Klebsiella pneumoniae M5a1: cloning, nucleotide sequence and expression; Binder F et al.; The structural gene encoding cyclodextrin-glycosyltransferase of Klebsiella pneumoniae strain M5a1 was cloned; it is expressed both in Escherichia coli and in K . pneumoniae and the gene product is secreted into the extracellular space . Determination of the nucleotide sequence revealed an open reading frame coding for a single polypeptide of 655 amino acid (aa) residues . The enzyme is synthesized as a precursor with an N-terminal signal peptide of 30 aa residues, which is proteolytically processed between two alanine residues during export . The primary structure of CGT bears homology with the sequences of amylases from both prokaryotic and eukaryotic origins.

Teratog Carcinog Mutagen, 1986, 6(6), 523 - 36
Developmental effects of chemicals and the heat shock response in Drosophila cells; Bournias-Vardiabasis N et al.; Exposure of prokaryotic and eukaryotic cells to heat shock (hyperthermia) or to a number of diverse environmental stresses such as teratogens, anoxia, and inhibitors of oxidative phosphorylation results in the enhanced synthesis of a number of proteins which have been previously referred to as heat shock proteins (hsps) . More recently, in view of the diverse types of agents that can induce these proteins, they have also been referred to as stress proteins . This phenomenon is one of the most basic regulatory mechanisms in living organisms . Exposure of Drosophila embryos, larvae, or pupae to these types of stresses also results in a variety of developmental abnormalities in the ensuing adult . Although the function(s) of these heat shock proteins has yet to be determined, they are widely thought to play an important role in cell survival and protection following some types of environmental stress . In our laboratory, we have developed an in vitro assay for detecting agents that act as teratogens, utilizing Drosophila embryonic cultures . Drosophila embryonic cells differentiate in vitro to a number of functional cell types including myotubes and ganglia . A number of drugs that have been shown to act as teratogens in mammals have also been found to inhibit muscle and/or neuron differentiation in Drosophila embryonic cultures . We have examined, by two-dimensional gel electrophoresis, the effects of such teratogens on protein synthesis in Drosophila embryonic cells . Inhibition of muscle and/or neuron differentiation correlates well with the induction of two proteins of about 20 kilodaltons . These are identical to two of the heat shock proteins (hsp 23, 22) as shown by electrophoretic mobilities and peptide mapping by partial proteolysis . Heat shock and other treatments such as exposure to some of the metal ions and ether induces the entire set of seven major heat shock proteins in the Drosophila embryonic cells . Dose-response studies of several teratogens show a correlation between the degree of inhibition of differentiation and the level of induction of hsps . Since heat shock proteins have been suggested as possibly serving a protecting role, our present studies are aimed at identifying the role of hsps in teratogenesis and investigating the differential regulation of heat shock genes in response to different external stimuli.

Comp Biochem Physiol B, 1986, 85(1), 93 - 9
Aspartate:2-oxoglutarate aminotransferase from Trichomonas vaginalis: comparison with pig heart cytoplasmic enzyme; Lowe PN et al.; The aspartate:2-oxoglutarate aminotransferase from the protozoon Trichomonas vaginalis exists as a mixture of sub-forms of identical Mr and amino acid composition, and of similar catalytic properties . The amino acid composition closely resembles that of aspartate aminotransferase from prokaryotic and vertebrate sources . Some molecular and catalytic properties of the T . vaginalis aspartate aminotransferase are compared with those of the cytoplasmic pig heart enzyme . A major difference is in the ability of the trichomonal enzyme to transaminate aromatic amino acids and 2-oxo acids . A range of inhibitors have been used to compare the active-site regions of the T . vaginalis and cytoplasmic pig heart aspartate aminotransferases.

Gene, 1986, 44(1), 1 - 10
The characterization of terminators of RNA transcription on IS30 and an analysis of their role in IS element-mediated polarity; Dalrymple B et al.; Using expression vectors carrying the lacUV5 or Pgal promoters and the galK gene, we have studied terminators of transcription on the prokaryotic mobile genetic element IS30 . The long open reading frame, ORF-A, of IS30 contains a relatively Rho-independent terminator, T30A, within its coding sequence . T30A terminates the majority of transcripts initiated at either an external promoter or the IS30-borne promoter P30A . No other terminator functions on this strand of IS30 (orientation left to right) . In the orientation right to left, the previously identified terminator T30C, which follows ORF-C, is Rho-independent . T30C together with T30D, a newly identified, strong, partially Rho-dependent terminator near the left end of IS30, permits less than 2% read-through from external promoters . Neither ORF-A nor ORF-C appears to be protected from transcription by external promoters . As a consequence of the internal terminators, the insertion of IS30 into an operon can be expected to reduce the expression of genes downstream of the site of insertion weakly for one orientation of IS30 and strongly for the other orientation.

Biosystems, 1986, 19(1), 23 - 44
Cell cycle modelling; Alberghina L et al.; Models able to describe the events of cellular growth and division and the dynamics of cell populations are useful for the understanding of functional control mechanisms and for the theoretical support for automated analysis of flow cytometric data and of cell volume distributions . This paper reports on models that we have developed with this aim for different kinds of cells . The models are composed by two subsystems: one describes the growth dynamics of RNA and protein, and the second accounts for DNA replication and cell division, and describe in a rather unitary frame the cell cycle of eukaryotic cells, like mammalian cells and yeast, and of prokaryotic cells . The model is also used to study the effects of various sources of variability on the statistical properties of cell populations, and we find that in microbial cells the main source of variability appears to be an inaccuracy of the molecular mechanism that monitors cell size . In normal mammalian cells another source of variability, that depends upon the interaction with growth factors which give competence, is apparent . An extended version of the model, which comprises also this additional variability, is presented and used to describe the properties of mammalian cell growth.

EMBO J, 1985 Dec 30, 4(13B), 3743 - 9
Effect of diadenosine tetraphosphate microinjection on heat shock protein synthesis in Xenopus laevis oocytes; Guedon G et al.; Bisnucleosides polyphosphates are thought to be chemical messengers signalling to the cell the onset of various stresses . Diadenosine tri- and tetraphosphates (respectively, Ap3A and Ap4A) accumulate in prokaryotic and eukaryotic cells under heat shock conditions, suggesting they could trigger the synthesis of heat shock proteins (hsps) . In this study, Ap4A, Ap3A and, as a control, Ap4 (adenosine tetraphosphate) were injected into Xenopus oocytes . Whereas none of these compounds is able to trigger the synthesis of hsps in the absence of hyperthermic treatment, nuclear microinjection of Ap4A after a mild heat shock specifically enhances the synthesis of the 70-kd hsp, which is involved in the regulation and possibly the termination of the heat shock response . The microinjection of Ap4A prior to the hyperthermic treatment results in a strong inhibition of hsps synthesis (with the exception of the 70-kd hsp) suggesting that Ap4A is involved in the regulation and/or termination of the heat shock response . Ap3A and Ap4 do not induce any detectable modification of hsps expression.

Nucleic Acids Res, 1985 Dec 20, 13(24), 8787 - 96
In vitro transcription initiation of the spinach chloroplast 16S rRNA gene at two tandem promoters; Lescure AM et al.; Two potential prokaryotic promoters, P1 and P2, are characterized 164 and 114 bp upstream of the spinach chloroplast 16S rRNA gene . The strengths of these promoters, calculated according to an homology score established for E . coli RNA-polymerase, are identical . Experiments performed with a Taq I-DNA fragment, containing 16 bp of the 16S rDNA and 243 bp upstream of the gene, give evidence that in vitro, E . coli RNA-polymerase starts transcription at these two promoters . These results are based on both the size of the transcripts and their nucleotide sequences . A possible regulation by differential control of these dual promoters is suggested . S1 mapping with RNAs extracted either from green or from etiolated spinach plants, indicates that, at these two steps of plastid development, transcription in vivo starts at P1 . Surprisingly only P2 appears to be conserved in the homologous sequences reported for maize, mustard and Spirodela.

EMBO J, 1985 Dec 16, 4(13A), 3375 - 83
Analysis of promoter regions for the spinach chloroplast rbcL, atpB and psbA genes; Gruissem W et al.; A promoter-deletion derivative of the spinach trnM2 gene was used for the identification and characterization of the promoter regions for the spinach chloroplast RuBisCo large subunit (rbcL), ATPase beta-subunit (atpB) and QB-polypeptide (psbA) genes . The DNA sequences 5' upstream from the transcriptional start sites of these genes share homology with the ctp1 and ctp2 arrangement found for the trnM2 transcription unit and the canonical Escherichia coli '-10' and '-35' promoter regions . Synthetic DNA fragments of approximately 40-bp regions, including the defined transcriptional start sites and proximal residues, from rbcL, atpB and psbA, were fused to the trnM2 deletion mutant 51 . The promoter-fusion constructs direct the correct transcription of tRNAMet2 in the chloroplast extract with distinct efficiencies . The ctp1- and ctp2-like elements in the trnM2, rbcL and psbA promoter regions can be interchanged to yield functional chimeric promoters of varying strengths . As a result, ctp1 sequences from atpB and psbA, trnM2 and rbcL, respectively, can be ordered TTGACA greater than TTGCTT greater than TTGCGC with respect to their intrinsic strengths . Single base pair changes were introduced into the ctp2-like element in the psbA promoter region . In analogy to similar base pair changes which lower promoter efficiency in E . coli, these mutations result in reduced transcription levels in the chloroplast extract . The data are consistent with a prokaryotic model for chloroplast promoter function.

J Biol Chem, 1985 Dec 15, 260(29), 15669 - 74
2H NMR investigation of the organization and dynamics of polyisoprenols in membranes; de Ropp JS et al.; The polyisoprenols (PIs) dolichol and undecaprenol function as chemical carriers of glycosyl residues in the membrane-directed synthesis of glycoconjugates in prokaryotic and eukaryotic cells . The molecular details of how these lipid cofactors function is unknown . Presented here are results of deuterium NMR investigations of site specifically 2H-labeled PIs incorporated into model membranes . To complement previous omega-terminal PI labeling schemes, a simple synthesis of head group 2H-labeled PIs is presented in which a PI alcohol is esterified with deuterated acetyl chloride . The 2H-labeled PIs, when incorporated into multilamellar membranes composed of phosphatidylcholine, gave rise to 2H NMR powder patterns interpretable in terms of quadrupole splittings (delta vQ) and spin-lattice relaxation times (T1s) . Pure isomers of head group 2H-labeled geraniol (C10) and solanesol (C45) gave rise to single splittings while farnesol (C15) gave rise to two sets of splittings due to cis-trans isomerization at the polar terminal double bond . Membranes containing C45 solanesol exhibited a large isotropic component, indicative of limited partitioning of this poly trans PI into the membrane . T1 measurements revealed high rates of motion for PIs relative to cholesterol in similar membrane hosts and revealed correlation times close to the fatty acyl methyl termini in phosphatidylcholine . The smaller PIs showed higher rates of motion but the T1s of head and tail labels were similar . These data indicate that both ends of the esterified PI molecules see similar environments, probably in the bilayer interior, and suggest that the esterified PIs studied here do not appear to adopt a conventional head group-at-interface orientation of lipids within the bilayer.

Nucleic Acids Res, 1985 Dec 9, 13(23), 8531 - 41
Sequence organization of repetitive elements in the flanking regions of the chloroplast ribosomal unit of Chlamydomonas reinhardii; Schneider M et al.; The flanking regions and the end of the chloroplast ribosomal unit of Chlamydomonas reinhardii have been sequenced . The upstream region of the ribosomal unit contains three open reading frames coding for 111, 117 and 124 amino acids, respectively . The latter polypeptide is partially related to the ribosomal protein L16 of E . coli . Two of the open reading frames overlap each other and are oriented in opposite direction . The region between these open reading frames and the 5' end of the 16S rRNA gene contains numerous short direct and inverted repeats which can be folded into large stem-loop structures . Sequence elements that resemble prokaryotic promoters are found in the same region . Several of the repeated elements are distributed throughout the non-coding regions of the chloroplast inverted repeat . Sequence comparison between the 5S rRNA and its gene does not reveal any significant sequence heterogeneity between the chloroplast 5S rRNA genes.

J Biol Chem, 1985 Dec 5, 260(28), 14897 - 900
Euglena gracilis chloroplast initiation factor 2 . Identification and initial characterization; Gold JC et al.; The chloroplast protein synthesis factor responsible for the binding of fMet-tRNAMeti to chloroplast 30 S ribosomal subunits (IF-2chl) has been identified in whole cell extracts of Euglena gracilis . The IF-2chl activity is present in considerably higher amounts in extracts of light-grown cells than in extracts of dark-grown cells . About 90% of this activity is found in the postribosomal supernatant of the cell . Chromatography on phosphocellulose results in the partial purification of IF-2chl and separates the chloroplast factor from the cytoplasmic factor eIF-2A . The binding of fMet-tRNAMeti to chloroplast 30 S subunits is message-dependent as observed for prokaryotic systems . In addition, GTP stimulates the IF-2chl-dependent reaction 3-fold . The binding reaction shows broad monovalent and divalent cation optima . The activity of IF-2chl is stimulated 2-fold by the addition of either Escherichia coli IF-1 or IF-3, and 4-fold by the inclusion of both factors . Chloroplast IF-2 is quite active on the homologous 30 S ribosomal subunits but shows little activity on E . coli 30 S or wheat germ 40 S subunits.

Biochemistry, 1985 Dec 3, 24(25), 7033 - 7
Sequence dependence of the curvature of DNA: a test of the phasing hypothesis; Hagerman PJ; Certain DNA molecules derived from both prokaryotic and eukaryotic organisms display markedly abnormal electrophoretic behavior on polyacrylamide gels . These molecules share a common element of sequence which involves collections of A/T residues that are approximately in phase with the helix repeat . This sequence periodicity has led to the suggestion that such phasing is important in generating the abnormal behavior . We have demonstrated that such phasing is, in fact, essential, thus ruling out alternative models which invoke any form of isotropic or centrosymmetric flexibility as the source of the phenomenon . We have also shown that the abnormal behavior is not a simple consequence of marginal thermodynamic stability . The most plausible explanation for the observed behavior is that stable, local distortions of the helix axis result in macroscopic curvature when such distortions are propagated in phase with the helix repeat.

Mol Gen Mikrobiol Virusol, 1985 Dec, (12), 3 - 11
{The role of surface structures of recipient cells in bacterial conjugation}; Domaradskii IV; Previously, while studying conjugation process in bacteria, the main attention was given to donors of plasmids . The aim of this review is to show that recipient cells indeed play an important role in this process too . The main attention is given to the structure of recipient cell walls, which is necessary for the recognition of donor cell pili and the establishment of the contact between the cells, i . e . to the first steps of conjugation between gram-negative microorganisms . The evidence concerning the influence of recipient cell walls defects on transmission of some plasmids is presented . The significant role of defects in lipopolysaccharide structure and of the intrinsic features of protein components of the outer membrane is considered . To the author's mind, the diversity in the cell wall structure creates the "outer barriers" for the plasmid exchange between different bacteria . Like the "inner barriers" (restriction, transcription, mistakes, plasmid incompatibility, etc) these "outer barriers" seem to be crucial for genetic isolation of bacterial species . Postulating this process, the author puts forward the idea of applicability of the conception of the biological species to prokaryotes.

Cell, 1985 Dec, 43(3 Pt 2), 729 - 36
A eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor; Brent R et al.; We describe a new protein that binds to DNA and activates gene transcription in yeast . This protein, LexA-GAL4, is a hybrid of LexA, an Escherichia coli repressor protein, and GAL4, a Saccharomyces cerevisiae transcriptional activator . The hybrid protein, synthesized in yeast, activates transcription of a gene if and only if a lexA operator is present near the transcription start site . Thus, the DNA binding function of GAL4 can be replaced with that of a prokaryotic repressor without loss of the transcriptional activation function . These results suggest that DNA-bound LexA-GAL4 and DNA-bound GAL4 activate transcription by contacting other proteins.

Tsitologiia, 1985 Dec, 27(12), 1374 - 9
{Transformation of mice by a prokaryotic gene for dihydrofolate reductase}; Titomirov AV et al.; In mice obtained after microinjection into the male pronucleus of fertilized eggs of the plasmid, containing the bacterial gene of dihydrofolate reductase (DHFR), under the control of the early promotor of the simian virus 40 (SV40), an integration of the foreign DNA into the mouse genome is found . About 30% of the treated animals contain the integrated plasmid DNA sequences, i.e . are transgenic . In 2 of 7 mice, containing the introduced plasmid in their genome, the methotrexate-resistant DHFR activity is found in the kidney and spleen, which may be due to the expression of gene DHFR . The plasmid DNA sequences and the ability to synthesise the methotrexate-resistant enzyme DHFR are transmitted to the next generation of mice.

Biofizika, 1985 Nov-Dec, 30(6), 1016 - 21
{Characteristics of the stoichiometric regulation of glycolysis in prokaryotic cells . A model}; Ivanitskaia IuG et al.; A model is developed to investigate the steady state of the glycolytic system of procaryotic cells which have the hexose transport into the cell coupled with phosphoenolpyruvic acid-dependent phosphorylation . Analysis of the model shows that this phosphorylation results: 1) in stoichiometric connection between the rate of hexose consumption and the utilization of ATP and 2) the stoichiometric positive feedback . The latter can be the cause of bistability, oscillations and other nonlinear phenomena.

Dev Biol, 1985 Nov, 112(1), 194 - 202
Cell-cell interactions in developmental lysis of Myxococcus xanthus; Janssen GR et al.; The developmental events of sporulation and fruiting body formation in the prokaryote Myxococcus xanthus are preceded by a stage of massive cell death . Two phenotypically complementable strains of M . xanthus defective in developmental lysis were identified from a group of conditional sporulation mutants . Mixture of the two lysis groups resulted in full complementation of lysis, sporulation, and fruiting body formation; efficient sporulation was observed only in strain mixtures where lysis was complemented . We have identified a cell-free extract from developing cells that phenotypically complemented lysis, sporulation, and fruiting body formation in one group of mutants; the active component of this extract appeared to be tightly cell associated . The effect of the cell-free extract could be replaced by exogenously supplied glucosamine or mannosamine.

Cell, 1985 Nov, 43(1), 351 - 60
Dual functions of the signal peptide in protein transfer across the membrane; Coleman J et al.; Most secretory proteins in both prokaryotic and eukaryotic cells are synthesized from a precursor with an amino-terminal extension of 20 to 25 amino acid residues called a signal peptide . These signal peptides are removed during translocation of the secretory proteins across the membrane . When two precursor structures are fused, the internalized second signal peptide was found to exert two different roles, depending upon either the distance between the two signal peptides, or whether the first signal peptide functions cotranslationally or posttranslationally . One role is to function as the usual signal peptide to translocate the protein downstream of the internal signal peptide . The other role is to function as a stop-transfer signal to create a transmembrane protein with the second signal peptide anchoring the protein in the membrane.

Proc Natl Acad Sci U S A, 1985 Nov, 82(21), 7232 - 6
Molecular cloning and regulated expression of the human c-myc gene in Escherichia coli and Saccharomyces cerevisiae: comparison of the protein products; Miyamoto C et al.; mRNA from human HL-60 cells was used to prepare a cDNA library, from which two full-length clones that encompass the complete c-myc coding region were isolated . One clone, pM1-11, contains all three exons of human c-myc . The second clone, pM4-10, represents a relatively rare transcript that initiated in the first intron and includes the coding exons 2 and 3 . The cDNA insert in pM1-11 was used to express the human c-myc protein in both prokaryotic and eukaryotic cells . Insertion of the coding sequences in exons 2 and 3 into the appropriate expression vectors yielded detectable c-myc protein in Escherichia coli lacking the Lon protease and in Saccharomyces cerevisiae upon induction . The protein produced in E . coli has an apparent size of 60 kDa and appears to be unmodified, as it is identical in size to the protein synthesized in an in vitro system . In contrast, yeast cells synthesize two myc proteins, of 60 kDa and 62 kDa . The difference in apparent molecular mass between the two proteins appears to be due, in part, to phosphorylation . Subcellular fractionation of yeast cells showed that the c-myc phosphoprotein is located predominantly in the nuclear fraction.

Proc Natl Acad Sci U S A, 1985 Nov, 82(22), 7656 - 60
Genetic complementation of UV-induced DNA repair in Chinese hamster ovary cells by the denV gene of phage T4; Valerie K et al.; The denV gene of phage T4, encoding the pyrimidine dimer-specific DNA repair enzyme endonuclease V, has been introduced by DNA transfection into the UV-sensitive DNA repair-deficient Chinese hamster ovary (CHO) cell line UV5 . Transformants were first selected for resistance to the antibiotic G418 conferred by the neo gene from Tn5 carried by the same plasmid . A majority of the isolated G418-resistant UV5 clones also showed an increased resistance to 254-nm UV light . One clone, designated I-A1, was found to have an intermediate level of colony-forming ability after UV irradiation when compared to UV5 and wild-type AA8 cells . A Southern blot showed that I-A1 carries a single integrated intact copy of the denV gene . Alkaline sucrose gradients revealed a dose-dependent appearance of breaks in the DNA of I-A1 cells following UV-irradiation, while unirradiated cells did not exhibit any significant breaks . Analysis of DNA repair by isopycnic sedimentation showed that DNA excision repair by I-A1 was at least equal to the level of repair in AA8 cells . These results show that the prokaryotic denV gene can restore UV repair capabilities in vivo to CHO UV5 cells defective in repair of UV-induced damage.

Nucleic Acids Res, 1985 Oct 25, 13(20), 7223 - 36
Molecular cloning and nucleotide sequence of a developmentally regulated gene from the cyanobacterium Calothrix PCC 7601: a gas vesicle protein gene; Tandeau de Marsac N et al.; Since the gas vesicle protein (GVP) is highly conserved among the different gas-vacuolate prokaryotes, a 29-mer oligonucleotide corresponding to a portion of the Anabaena flos-aquae GVP gene was synthesized and used to isolate the GVP structural gene from Calothrix PCC 7601 (= Fremyella diplosiphon) . Gas vacuole production in this filamentous cyanobacterium is restricted to hormogonia which occur at a specific stage during the developmental cell cycle . The GVP gene (gvpA) was localized on a 709 bp HindIII-HincII fragment . Nucleotide sequence analysis revealed a 213 bp open reading frame whose deduced amino-acid sequence shows a very high homology with that of the Anabaena flos-aquae GVP . Assuming that the first methionine residue is proteolytically processed, the molecular mass of the Calothrix GVP is 7375 daltons . Sequences resembling the Escherichia coli consensus promoter were found upstream from the gvpA gene . The initiator codon of the gvpA gene is preceded by a polypurine sequence assumed to be the ribosome binding site . Southern hybridizations with a probe specific for the gvpA gene indicated that this gene is not plasmid-borne, and that another homologous gene is present in the Calothrix genome.

J Biol Chem, 1985 Oct 15, 260(23), 12695 - 9
CMP-NeuNAc:poly-alpha-2,8-sialosyl sialyltransferase and the biosynthesis of polysialosyl units in neural cell adhesion molecules; McCoy RD et al.; Prokaryotic derived probes that specifically recognize alpha-2,8-ketosidically linked polysialosyl units were developed to identify and study the temporal expression of these unique carbohydrate moieties in developing neural tissue (Vimr, E . R., McCoy, R . D., Vollger, H . F., Wilkison, N . C., and Troy, F . A . (1984) Proc . Natl . Acad . Sci . U.S.A . 81, 1971-1975) . These polysialosyl units cap N-linked oligosaccharides of the complex-type on neural cell adhesion molecules (N-CAM) . A Golgi-enriched fraction from 20-day-old fetal rat brain contains a membrane-associated sialyltransferase that catalyzes the incorporation of {14C}N-acetylneuraminic acid {( 14C}NeuNAc) from CMP-{14C} NeuNAc into polymeric products . At pH 6.0, 84 pmol of NeuNAc mg of protein-1 h-1 were incorporated . In sodium dodecyl sulfate-polyacrylamide gels, the major radiolabeled species migrated with a mobility expected for N-CAM . A bacteriophage-derived endoneuraminidase specific for polysialic acid was used to demonstrate that at least 20-30% of the {14C}NeuNAc was incorporated into alpha-2,8-linked polysialosyl units . This was confirmed by structural studies which showed that the endoneuraminidase-sensitive brain material consisted of multimers of sialic acid . The addition of a partially purified preparation of chick N-CAM to the membranous sialyltransferase stimulated sialic acid incorporation 3-fold . The product of this reaction was also sensitive to endoneuraminidase and contained alpha-2,8-linked polysialosyl chains, thus showing that N-CAM can serve as an exogenous acceptor for sialylation in vitro . Sialic acid incorporated into adult rat brain membranes was resistant to endoneuraminidase, indicating that the poly-alpha-2,8-sialosyl sialyltransferase activity is restricted to an early developmental epoch . It is recommended that the enzyme described here be designated CMP-NeuNAc:poly-alpha-2,8-sialosyl sialyltransferase and the trivial name poly-alpha-2,8-sialosyl sialyltransferase be adopted.

Nucleic Acids Res, 1985 Oct 11, 13(19), 6797 - 816
Poliovirus genome RNA hybridizes specifically to higher eukaryotic rRNAs; McClure MA et al.; The RNA genome of poliovirus hybridizes to 28S and 18S rRNAs of higher eukaryotes under stringent conditions . The hybridization detected by Northern blot analyses is specific since little or no signal was detected for yeast or prokaryotic rRNAs or other major cellular RNAs . Southern blot analysis of DNA clones of mouse rRNA genes leads us to conclude that several regions of 28S rRNA, and at least one region in 18S rRNA, are involved in the hybridization to polio RNA, and that G/C regions are not responsible for this phenomenon . We have precisely mapped one of these hybridizing regions in both molecules . Computer analysis confirms that extensive intermolecular base-pairing (81 out of 104 contiguous bases in the rRNA strand) could be responsible for this one particular site of interaction (polio genome, bases 5075-5250; 28S rRNA, bases 1097-1200) . We discuss the possible functional and/or evolutionary significance of this novel type of interaction.

J Theor Biol, 1985 Oct 7, 116(3), 427 - 33
Eukaryotic oligomer frequencies are correlated with certain DNA helical parameters; Lennon GG et al.; Nucleotide sequences were converted into purine (R)-pyrimidine (Y) series and divided into several groups, embracing higher and lower organisms . The frequencies of R-Y doublets, triplets and quartets in each were calculated . Whereas eukaryotes uniformly show RR + YY greater than RY + YR, in bacteria and phage no such relationship is observed . The triplet and quartet patterns in higher organisms differ from those seen in prokaryotes . In the higher organisms a correlation is observed between the frequencies of triplets and quartets and some DNA structural parameters . Specifically, the most frequent triplets are those with minimal torsion angle deviations from a regular B-DNA . The most frequent quartets are those with minimal roll angle deviations . No such correlations are observed in prokaryotes . We therefore propose that in eukaryotic DNA, tight, smooth packaging imposes sequence constraints.

Nature, 1985 Oct 31-Nov 6, 317(6040), 822 - 4
Negative control at a distance mediates catabolite repression in yeast; Struhl K; In prokaryotic organisms, the control of gene expression is mediated by regulatory proteins that activate or repress transcription . However, the molecular mechanisms of positive and negative control are different . In terms of negative control, repressor proteins bind to sites located within the promoter region and as a consequence sterically interfere with functional binding by RNA polymerase . Here, I examine the properties of a regulatory sequence that specifies catabolite (glucose) repression in the yeast Saccharomyces cerevisiae . Specifically, a DNA segment containing this regulatory site was fused upstream of the intact his3 promoter region and structural gene at several locations . Normally, his3 expression in these derivatives occurs at a basal level which can be induced by conditions of amino-acid starvation . However, in glucose medium, the catabolite regulatory sequence overrides the normal his3 promoter elements and reduces transcription both in normal and starvation conditions . The implication from these results is that in contrast to catabolite repression in Escherichia coli, which is mediated by catabolite-activating protein (CAP), catabolite repression in yeast occurs by a negative control mechanism involving a putative repressor protein . The observation that this regulatory site exerts its repressing effects even when located upstream of an intact promoter region suggests that repression in yeast is not mediated by steric interference between regulatory proteins and the transcriptional apparatus.

Biol Chem Hoppe Seyler, 1985 Oct, 366(10), 963 - 9
Purification and properties of an FAD-containing NADH oxidase from Mycoplasma capricolum; Klomkes M et al.; From the prokaryotic microorganism Mycoplasma capricolum an FAD-containing NADH oxidase has been purified by preparative FPLC to homogeneity, as judged by polyacrylamide gel electrophoresis . The apparent molecular mass of the enzyme was found to be 72.5 kDa, with an isoelectric point of 5.2, and no detectable subunits . No iron, copper, manganese or molybdenium could be detected . On the basis of a minimum molecular mass of 72.5 kDa a ratio of FAD/protein of 1:1 could be derived . Its amino-acid composition, the light absorption and the fluorescence spectra are presented.

Mutat Res, 1985 Oct, 144(2), 99 - 106
Studies on germinal effects of quercetin, a naturally occurring flavonoid; Aravindakshan M et al.; Quercetin (3,3',4',5,7-pentahydroxyflavone) is one of the most widely occurring flavonoids ingested by man in food . It has been shown to be mutagenic in prokaryotes as well as in in vitro mammalian cell lines . In view of the unavoidability of ingesting it via a normal diet, there is a need to assess the potential genetic risk to man, due to flavonoid ingestion, using whole animal assays . Dominant lethal studies have been carried out in adult Swiss male mice and Wistar male rats to investigate the germinal effects of quercetin . Adult Swiss males were treated with 200, 300 or 400 mg/kg of quercetin dissolved in 60% dimethyl sulfoxide, intraperitoneally . In the rat study, 200 and 300 mg/kg of quercetin were used . Individually caged males were paired with untreated females for a week and six sequential matings were carried out . Two independent experiments in mice and a single experiment in rats constituted the study . Females were evaluated for inducted dominant lethality during the midterm of pregnancy . At 200 mg/kg dose of quercetin, there were no significant differences between control and the test group in pregnancy, total or live implantations in mice or rats during the whole test period that could be attributed to the flavonoid exposure . In mice at 300 and 400 mg/kg of quercetin, there was a profound reduction in fertility of the males during all six matings . The number of total and live implantations also decreased, particularly at the 400 mg/kg dose, although the sample size was too small to be statistically significant . Contrary to this, the rat study did not show any impairment of fertility, nor was there any substantial suppression of total and live implantations at the highest (300 mg/kg) dose tested . There were no significant differences between control and treated groups with regard to the number of dead implantations at any dosage level at any stage of the study in mice and rats . Thus, no post-implantation losses--a reliable measure of dominant lethal mutations--were induced by quercetin in mice or rats . The loss of fertility could be due to germinal cytotoxicity, oligospermia or impairment of fertilizing ability of the treated animals.(ABSTRACT TRUNCATED AT 400 WORDS)

J Virol Methods, 1985 Oct, 12(1-2), 111 - 33
Biochemical and electron microscopic studies of the transcription of vaccinia DNA by RNA polymerase from Escherichia coli: localization and characterization of transcriptional complexes; Esteban M et al.; We used the prokaryotic Escherichia coli RNA polymerase to determine if vaccinia DNA might provide recognition sites for the bacterial binding and initiation . Electron microscopic studies of the interaction of E . coli RNA polymerase with vaccinia DNA and molecular hybridization analysis of the transcription products formed after 3 or 5 min of in vitro incubation showed that: there were 30-40 sites on the template where the polymerase could bind and initiate cRNA synthesis; the entire coding capacity of the genome was utilized for cRNA synthesis; transcription was asymmetric; cRNA molecules were similar in size to the transcripts synthesized by the vaccinia virus RNA polymerase in vitro and in vivo; cRNA contains sequences in common with 'pre-early', 'early', and 'late' in vivo RNA; 'self-annealing' of cRNA in the presence or absence of RNA synthesized in vitro by the virion associated RNA polymerase showed that less than 1% dsRNA product could be detected suggesting that initially the same strand(s) was copied by the viral and bacterial enzymes; no differences in the frequency with which sequences represented in the Hind III fragments of vaccinia DNA were transcripted with time of in vitro incubation could be detected . These findings strongly suggest that the bacterial enzyme might recognize truly viral promotors . With extended in vitro incubations of the E . coli RNA polymerase with vaccinia DNA the control of transcription was found to diminish . This was correlated with an increase in the size of the transcripts and the synthesis of significant amounts of self-complementary RNA, indicating that symmetrical transcription was occurring . The dsRNA species recovered after self-annealing the cRNA from a 30 min in vitro reaction mixture were found to contain sequences which hybridized to some portion of all the Hind III restriction fragments of vaccinia DNA . The methods described here might be useful for the localization and characterization of promotor sequences in the genome of vaccinia virus, as well as for studies on sequence conservation between members of the Poxvirus genus.

Genetics, 1985 Oct, 111(2), 375 - 88
Homologous recombination between autonomously replicating plasmids in mammalian cells; Ayares D et al.; The ability of autonomously replicating plasmids to recombine in mammalian cells was investigated . Two deletion plasmids of the eukaryotic-prokaryotic shuttle vector pSV2neo were cotransfected into transformed monkey COS cells . Examination of the low molecular weight DNA isolated after 48 hr of incubation revealed that recombination between the plasmids had occurred . The DNA was also used to transform recA- E . coli . Yield of neoR colonies signified homologous recombination . Examination of the plasmid DNA from these colonies confirmed this view . Double-strand breaks in one or both of the input plasmids at the sites of deletion resulted in an enhancement of recombination frequency . The recombination process yielded monomeric and dimeric molecules . Examination of these molecules revealed that reciprocal recombination as well as gene conversion events were involved in the generation of plasmids bearing an intact neo gene . The COS cell system we describe is analogous to study of bacteriophage recombination and yeast random-spore analysis.

Proc Natl Acad Sci U S A, 1985 Oct, 82(20), 7025 - 9
cDNA of the immunoglobulin kappa chain of an Epstein-Barr virus-transformed human lymphoid cell line: partial sequence determination and bacterial expression; Morin JW et al.; We report the isolation, nucleotide sequence determination, and bacterial expression of a partial cDNA for the immunoglobulin kappa chain from the Epstein-Barr virus-transformed human lymphoid cell line GM131 . The cDNA, cloned in pBR322 by use of oligo(dG) X oligo(dC) tails, yields two Pst I fragments of 250 and 600 base pairs (bp) . Various restriction enzyme fragments of the cDNA were subcloned in the vectors M13 mp10 and M13 mp11 for sequence analysis . As a result of instability of the 250-bp M13 subclones, the base sequence of the 250-bp Pst I fragment could not be determined . The 600-bp Pst I fragment contains coding sequences for part of the variable (V) region (residues 78-95) and all of the joining (J) (residues 96-108) and constant (C) regions (residues 109-212) and extends 148 bp into the 3' flanking region . Although the C- and 3'-flanking-region sequences are identical to germ-line sequences, the J-region sequence does not correspond to any of the five human germ-line J regions . The sequence is most similar to that of J4, with three base changes resulting in one silent mutation and two amino acid substitutions, at residues 103 (Lys----Tyr) and 106 (Ile---Met) . The silent mutation appears to be the result of RNA splicing between the J and the C regions . The V-region sequence differs from published V-region germ-line sequences at several codons and from the more common amino acid sequences at two positions, residues 91 and 93 . At these positions, histidine residues are found in place of the more common tyrosine and serine, respectively . None of the four amino acid substitutions observed for the GM131 kappa-chain are unique, suggesting that the changes, which most likely contribute to antigenic specificity, are compatible with antibody structure and function . The 600-bp Pst I fragment was subcloned in two prokaryotic expression vectors, pATH11 and pUC8 . In both instances, a kappa-chain fusion protein detectable by immunoblotting was produced.

Philos Trans R Soc Lond B Biol Sci, 1985 Sep 12, 310(1144), 291 - 6
The newer vaccines; Brown F; Vaccination is a powerful weapon in the control of animal diseases and many highly successful vaccines have been developed, particularly for virus diseases such as rinderpest, foot-and-mouth disease and Newcastle disease . Despite their extraordinary success, however, there are sufficient problems associated with their production and quality control to warrant a re-examination of the methods in current use . Dissection of virus particles into biologically active fragments has shown that their immunizing activity is usually carried on a single protein . With the identification of the genes coding for these individual proteins it is now possible to express these immunogens in both prokaryotic and eukaryotic cells . Moreover, the immunogenic sites of some of these proteins have been identified, synthesized in E . coli cells or by chemical methods and shown to possess immunizing activity . It is too early to put a time-scale on the commercial availability of the new vaccines . However, the potential advantages of such products over conventional vaccines has led to considerable effort in this field of research and progress has been so rapid that new vaccines could be available within the next few years.

Eur J Biochem, 1985 Sep 2, 151(2), 405 - 10
Prokaryotic triterpenoids . New bacteriohopanetetrol cyclitol ethers from the methylotrophic bacterium Methylobacterium organophilum; Renoux JM et al.; Together with bacteriohopanetetrol, which is identical to bacteriohopanetetrol isolated from Bacillus acidocaldarius, three other bacteriohopane derivatives were isolated from the methylotrophic bacterium Methylobacterium organophilum . Three different kinds of polar moieties were found linked to the C-35 hydroxyl group of bacteriohopanetetrol: glucosamine linked through a glycosidic bond and two new polyhydroxylated methylcyclopentanoids differing one from each other by the presence of an amino or a guanidino group and linked through an ether bond . The significance of the presence of these compounds in terms of membrane reinforcement and DNA/triterpenoid interactions is discussed.

EMBO J, 1985 Sep, 4(9), 2385 - 8
Structural details of the binding of guanosine diphosphate to elongation factor Tu from E . coli as studied by X-ray crystallography; la Cour TF et al.; Structural details of the guanosine diphosphate binding to a modified form of elongation factor Tu from Escherichia coli, resulting from X-ray crystallographic studies, are reported . The protein elements that take part in the nucleotide binding are located in four loops connecting beta-strands with alpha-helices . These loops correspond to regions in primary sequences which show a high degree of homology when compared with other prokaryotic and eukaryotic elongation factors and initiation factor 2.

Cell, 1985 Sep, 42(2), 599 - 610
Extensive homology among the largest subunits of eukaryotic and prokaryotic RNA polymerases; Allison LA et al.; We have determined the nucleotide sequence of two yeast RNA polymerase genes, RPO21 and RPO31, which encode the largest subunits of RNA polymerases II and III, respectively . The RPO21 and RPO31 sequences are homologous to each other, to the sequence of the largest subunit of E . coli RNA polymerase, and to sequences in the putative DNA-binding domain of E . coli DNA polymerase I . RPO21 has an unusual heptapeptide sequence tandemly repeated 26 times at its C-terminus; this sequence is conserved in the RNA polymerase II of higher eukaryotes and may play an important role in polymerase II-mediated transcription . Since eukaryotic and prokaryotic RNA polymerases appear to have evolved from a common ancestral polymerase, other features of the transcription process may also be evolutionarily conserved.

J Antibiot (Tokyo), 1985 Sep, 38(9), 1237 - 45
The mechanism of action of myxovalargin A, a peptide antibiotic from Myxococcus fulvus; Irschik H et al.; Myxovalargin A has two modes of action . At low concentrations (below 1 microgram/ml) it inhibits bacterial protein synthesis specifically and instantaneously . In vitro experiments suggest that it interferes with the binding of aminoacyl tRNA to the A site of the ribosome . At higher concentrations (above 5 micrograms/ml), or upon prolonged incubation, the antibiotic damages cell membranes . This leads to secondary effects, like decreased O2 consumption or instant break down of RNA synthesis, and may be the reason for the irreversibility of the antibiotic action . The membrane effect is not restricted to prokaryotes and may explain the high toxicity of the compound for higher organisms.

Vaccine, 1985 Sep, 3(3 Suppl), 165 - 71
Biological and immunological properties of haemagglutinin and neuraminidase expressed from cloned cDNAs in prokaryotic and eukaryotic cells; Nayak DP et al.; To study the biological and immunological properties of influenza virus surface glycoproteins, cDNA copies of the haemagglutinin (HA) and the neuraminidase (NA) genes of A/WSN/33 influenza virus were cloned and expressed in prokaryotic and eukaryotic cells . In Escherichia coli, maximum expression of HA is obtained only as a fusion protein in which the NH2-terminal portion is provided by a bacterial protein (i.e . beta gal or trpLE') . The HA expressed in bacteria (bacterial HA) is recognized by polyclonal anti-WSN antibodies but not by neutralizing monoclonal antibodies . The antibodies made against the bacterial HA bind to the detergent-treated viral HA, intact virus and live influenza infected cells, but fail to show either haemagglutination inhibition (HI) or virus neutralization . These results suggest that the three-dimensional structure as well as the antigenic epitopes of the bacterial HA are different from that of native viral HA . HA, expressed from cDNA in cultured animal cells, is shown to possess the structural features of the native viral HA . It is glycosylated, transported to the apical domain of the plasma membrane of polarized cells, causes haemadsorption and can induce cell to cell fusion at low pH after proteolytic cleavage . An attempt was made to define the structural features of HA required for sorting and directional transport by making chimeras with vesicular stomatitis virus G (VSV G) proteins either by switching the amino terminus or the carboxy terminus of HA with that of VSV G . These chimeric proteins were translocated across the rough endoplasmic reticulum (RER) but were blocked in transport between the RER and cell membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1985 Aug 27, 24(18), 4766 - 71
Differential modulation by spermidine of reactions catalyzed by type 1 prokaryotic and eukaryotic topoisomerases; Srivenugopal KS et al.; In the absence of DNA aggregation, spermidine inhibited the relaxation of negatively supercoiled DNA by Escherichia coli topoisomerase I at concentrations of the polyamine normally found intracellularly . Spermidine also curtailed the cleavage of negatively supercoiled ColE1 DNA by the enzyme in the absence of Mg2+ . On the contrary, knotting of M13 single-stranded DNA circles catalyzed by topoisomerase I was stimulated by the polyamine . Relaxation of supercoiled DNA by eukaryotic type 1 topoisomerases, such as calf thymus topoisomerase I and wheat germ topoisomerase, was significantly stimulated by spermidine in the same range of concentrations that inhibited the prokaryotic enzyme . In reactions catalyzed by S1 nuclease, the polyamine enhanced the digestion of single-stranded DNA and inhibited the nicking of negatively supercoiled DNA . These results suggest that spermidine modifies the supercoiled duplex substrate in these reactions by modulating the degree of single strandedness.

Eur J Biochem, 1985 Aug 15, 151(1), 101 - 10
Leucyl-tRNA and lysyl-tRNA synthetases, derived from the high-Mr complex of sheep liver, are hydrophobic proteins; Cirakoglu B et al.; The leucyl-tRNA and lysyl-tRNA synthetase components of the multienzyme complex from sheep liver were selectively dissociated by hydrophobic interaction chromatography on hexyl-agarose and purified to homogeneity . Conservation of activities during the purification required the presence of Triton X-100 . The homogeneous enzymes corresponded to a monomer of Mr 129000 and a dimer of Mr 2 X 79000, respectively . Both were strongly adsorbed to the hydrophobic support phenyl-Sepharose, in conditions where the corresponding purified enzymes from yeast and Escherichia coli were not bound . Moreover, like the corresponding enzymes from yeast but unlike those of prokaryotic origin, the purified leucyl-tRNA and lysyl-tRNA synthetases derived from the complex displayed affinity for polyanionic supports . It is shown that proteolytic conversion of lysyl-tRNA synthetase to a fully active dimer of Mr 2 X 64000, leads to loss of both the hydrophobic and the polyanion-binding properties . These results support the view that each subunit of lysyl-tRNA synthetase is composed of a major catalytic domain, similar in size to the subunit of the prokaryotic enzyme, contiguous to a chain extension which carries both cationic charges and hydrophobic residues . The implications of these findings on the structural organization of the complex are discussed in relation to its other known properties.

EMBO J, 1985 Aug, 4(8), 2101 - 7
Yeast ribosomal protein L25 binds to an evolutionary conserved site on yeast 26S and E . coli 23S rRNA; el-Baradi TT et al.; The binding site of the yeast 60S ribosomal subunit protein L25 on 26S rRNA was determined by RNase protection experiments . The fragments protected by L25 originate from a distinct substructure within domain IV of the rRNA, encompassing nucleotides 1465-1632 and 1811-1861 . The protected fragments are able to rebind to L25 showing that they constitute the complete protein binding site . This binding site is remarkably conserved in all 23/26/28S rRNAs sequenced to date including Escherichia coli 23S rRNA . In fact heterologous complexes between L25 and E . coli 23S rRNA could be formed and RNase protection studies on these complexes demonstrated that L25 indeed recognizes the conserved structure . Strikingly the L25 binding site on 23S rRNA is virtually identical to the previously identified binding site of E . coli ribosomal protein EL23 . Therefore EL23 is likely to be the prokaryotic counterpart of L25 in spite of the limited homology displayed by the amino acid sequences of the two proteins.

Nucleic Acids Res, 1985 Jul 25, 13(14), 5269 - 82
Purification and characterization of DNA polymerase from the archaebacterium Sulfolobus acidocaldarius; Klimczak LJ et al.; DNA polymerase has been purified about 25,000-fold from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius . On SDS-PAGE the enzyme was observed to have a molecular weight of 100 kDa and to be about 90% pure . The native molecular weight was 108 kDa indicating that the enzyme is composed of a single polypeptide . Activity gel analysis showed an active polypeptide of about 100 kDa . Under conditions promoting proteolysis this polypeptide was degraded to a slightly smaller form of 98 kDa . The enzyme has been characterized in respect to optimal assay conditions, template specificity, sensitivity to inhibitors and associated nuclease activities . The high temperature optimum of 65 degrees C should be emphasized . No substantial similarities have been found with other prokaryotic and eukaryotic DNA polymerases, although the enzyme bears certain resemblances to prokaryotic non-replicative polymerases.

Nature, 1985 Jul 25-31, 316(6026), 359 - 61
A transposon-like element in human DNA; Paulson KE et al.; Mobile genetic elements have been reported in prokaryotes, plants, yeast and Drosophila . The only transposon-like sequences reported for mammalian organisms are closely related to retroviruses, although undoubtedly other transposon families exist within the mammalian genome . Although mobile genetic elements can only be identified as such if their mobility can be demonstrated in existing populations, transposon and transposon-like elements share several common biochemical and structural features . Here we demonstrate that a repetitive human sequence has many of the diagnostic features of transposable elements . This 2.3-kilobase (kb) transposon-like element contains two flanking long terminal repeat (LTR)-like 350-base pair (bp) repetitive sequences, each of which begins with the sequence 5' TG.. . and ends with ...CA 3' . The transposon-like element is bounded by 5-bp direct repeats . Discrete-length polyadenylated transcripts from HeLa cells are homologous to the transposon-like element . Members of this transposon-like family are found in extrachromosomal circular DNA molecules.

FEBS Lett, 1985 Jul 22, 187(1), 11 - 5
Two functional domains conserved in major and alternate bacterial sigma factors; Stragier P et al.; Sequences of the sigma factors of Escherichia coli and Bacillus subtilis were aligned with the sequences of two sigma-like proteins, HtpR, involved in the expression of heat-shock genes in E . coli, and SpoIIG, necessary for endospore formation in B . subtilis . An internal region is highly conserved in the four proteins and is proposed to be involved in binding of sigma factors to core RNA polymerase . The carboxy-terminal part of the four proteins presents the characteristic structure found in several prokaryotic DNA-binding proteins and is proposed to be involved in promoter recognition.

J Theor Biol, 1985 Jul 21, 115(2), 179 - 89
Large helical conformational deviations from ideal B-DNA and prokaryotic regulatory sites; Nussinov R; The variations in several double helical DNA angular parameters have been calculated along about 60 bacterial and phage sequences, each several hundreds nucleotides long . Regions of large geometric irregularities are found at, or in the vicinity of, regulatory protein recognition sites . Based on these extensive computations I suggest that these structurally "wrinkled" regions facilitate the first stage of the recognition process.

Science, 1985 Jul 19, 229(4710), 275 - 7
Presence of laminin receptors in Staphylococcus aureus; Lopes JD et al.; A characteristic feature of infection by Staphylococcus aureus is bloodstream invasion and widespread metastatic abscess formation . The ability to extravasate, which entails crossing the vascular basement membrane, appears to be critical for the organism's pathogenicity . Extravasation by normal and neoplastic mammalian cells has been correlated with the presence of specific cell surface receptors for the basement membrane glycoprotein laminin . Similar laminin receptors were found in Staphylococcus aureus but not in Staphylococcus epidermidis, a noninvasive pathogen . There were about 100 binding sites per cell, with an apparent binding affinity of 2.9 nanomolar . The molecular weight of the receptor was 50,000 and pI was 4.2 . Eukaryotic laminin receptors were visualized by means of the binding of S . aureus in the presence of laminin . Prokaryotic and eukaryotic invasive cells might utilize similar, if not identical, mechanisms for invasion.

J Mol Biol, 1985 Jul 5, 184(1), 99 - 105
Signal sequences . The limits of variation; von Heijne G; Variations in length and composition of the charged N-terminal, central hydrophobic and polar C-terminal regions in a large sample of signal sequences have been mapped, both as a function of the overall length of the sequence, and in an absolute sense, i.e . various "extremes" have been sought . The results show subtle differences between eukaryotic and prokaryotic sequences, but the general impression of signal sequences as being highly variable is reinforced . Criteria for a "minimal" signal sequence are suggested and discussed.

Eur J Biochem, 1985 Jul 1, 150(1), 35 - 9
Prokaryotic triterpenoids . 3 . The biosynthesis of 2 beta-methylhopanoids and 3 beta-methylhopanoids of Methylobacterium organophilum and Acetobacter pasteurianus ssp . pasteurianus; Zundel M et al.; The incorporation of L-{methyl-3H,14C}methionine or L-(methyl-2H3)methionine into 2 beta-methyldiplopterol of Methylobacterium organophilum and various 3 beta-methylhopanoids of Acetobacter pasteurianus ssp . pasteurianus showed that all three hydrogen atoms of the transferred methyl group are retained in the triterpenoids . These methylations are compatible with a methylation substrate such as a delta 2-hopanoid in the case of the 2 beta-methylhopanoid biosynthesis and of a delta 2-hopanoid or squalene in the case of the formation of 3 beta-methylhopanoids . The intervention of intermediates possessing an exomethylene group or a cyclopropane ring is excluded.

Eur J Biochem, 1985 Jul 1, 150(1), 29 - 34
Prokaryotic triterpenoids . 2 . 2 beta-Methylhopanoids from Methylobacterium organophilum and Nostoc muscorum, a new series of prokaryotic triterpenoids; Bisseret P et al.; 2 beta-Methylhopanoids, a new series of triterpenoids was identified from two prokaryotes . 2 beta-Methyldiplopterol was isolated from the methylotrophic bacterium Methylobacterium organophilum, and three different 2 beta-methylbacteriohopanepolyols from the cyanobacterium Nostoc muscorum . The structures of these compounds was deduced by direct comparison with 2 beta-methyldiplopterol synthesized from 22-hydroxyhopan-3-one.

Eur J Biochem, 1985 Jul 1, 150(1), 23 - 7
Prokaryotic triterpenoids . 1 . 3 beta-Methylhopanoids from Acetobacter species and Methylococcus capsulatus; Zundel M et al.; 3 beta-Methylbacteriohopanepolyol derivatives were isolated from three bacteria, Acetobacter pasteurianus ssp . pasteurianus, Methylococcus capsulatus and Nostoc muscorum, and identified by spectroscopic methods and direct comparison with 3 beta-methyldiplopterol and 3 beta-methylhopan-29-ol synthesized from 22-hydroxyhopan-3-one . The 3 beta-methylhopanoid content of A . pasteurianus ssp . pasteurianus could be dramatically increased (up to 60% of the total hopanoid content) by addition of L-methionine, the actual methyl donor, to the culture medium.

Mutat Res, 1985 Jul, 146(1), 71 - 7
Microinjected photoreactivating enzymes from Anacystis and Saccharomyces monomerize dimers in chromatin of human cells; Zwetsloot JC et al.; Photoreactivating enzymes (PRE) from the yeast Saccharomyces cerevisiae and the cyanobacterium Anacystis nidulans have been injected into the cytoplasm of repair-proficient human fibroblasts in culture . After administration of photoreactivation light, PRE-injected cells displayed a significantly lower level of UV-induced unscheduled DNA synthesis (UDS) than non-injected cells . This indicates that monomerization of the UV-induced pyrimidine dimers in the mammalian chromatin had occurred as a result of photoreactivation by the injected PRE at the expense of repair by the endogenous excision pathway . Purified PRE from yeast is able to reduce UDS to 20-25% of the UDS found in non-injected cells, whereas the in vitro more active PRE from A . nidulans gives a reduction to only 70% . This suggests that the eukaryotic enzyme is more efficient in the removal of pyrimidine dimers from mammalian chromatin than its equivalent purified from the prokaryote A . nidulans.

EMBO J, 1985 Jul, 4(7), 1637 - 44
Identification and mutational analysis of the promoter for a spinach chloroplast transfer RNA gene; Gruissem W et al.; A transcription extract from purified spinach chloroplast was used to test chloroplast DNA sequences for their function as promoter elements . Chloroplast tRNA genes are correctly transcribed in the extract by a soluble RNA polymerase, and precursor molecules are processed into mature tRNAs . Transcription of the spinach chloroplast tRNA2Met gene (trnM2) in vitro requires 5' upstream DNA sequences . Deletion of 5' DNA sequences with exonuclease Bal31 was used to establish the 5' boundary of the promoter region . This boundary is part of a DNA sequence with partial homology to the prokaryotic -35 region . Seventeen base pairs downstream from this sequence a DNA sequence occurs which is homologous to the prokaryotic -10 region . We used synthetic oligonucleotides fused to trnM2 5' deletion mutants to create insertions, deletions and base substitutions in these regions . Internal deletion mutants demonstrated that the -10 promoter element is also required for transcription in vitro . The arrangement of DNA sequences recognised by the chloroplast RNA polymerase resembles the prokaryotic promoter organization.

Biochemistry, 1985 Jun 18, 24(13), 3263 - 7
Stereochemistry of lysine formation by meso-diaminopimelate decarboxylase from wheat germ: use of 1H-13C NMR shift correlation to detect stereospecific deuterium labeling; Kelland JG et al.; The stereochemical course of the wheat germ meso-diaminopimelate (DAP) decarboxylase reaction is compared to that of the decarboxylase isolated from Bacillus sphaericus, which has been reported to proceed with an unusual inversion of configuration {Asada, Y., Tanizawa, K., Sawada, S., Suzuki, T., Misono, H., & Soda, K . (1981) Biochemistry 20, 6881-6886} . Reaction of each enzyme with either unlabeled diaminopimelic acid in D2O or {2,6-2H2}diaminopimelic acid in H2O gave stereospecifically deuterium-labeled lysine samples that were derivatized with (-)-camphanoyl chloride and diazomethane . Analysis by two-dimensional 1H-13C heteronuclear NMR shift correlation spectroscopy with 2H decoupling confirmed the stereochemistry of the B . sphaericus enzyme reaction and showed that the eukaryotic wheat germ meso-DAP decarboxylase also operates with inversion of configuration . This suggests similar mechanisms for the prokaryotic and eukaryotic enzymes and contrasts the retention mode observed with other pyridoxal phosphate dependent alpha-decarboxylases.

Nature, 1985 Jun 13-19, 315(6020), 601 - 3
A novel immune system against bacteriophage infection using complementary RNA (micRNA); Coleman J et al.; The "operon' theory of gene regulation, in which protein repressor molecules bind to the operator site of a gene to prevent its transcription, is now well established . Recently, however, cases have been discovered in which gene expression is regulated by complementary RNA molecules that are able to bind to the transcripts of particular genes and consequently prevent their translation . For example, the synthesis of OmpF protein (a major outer membrane protein) in Escherichia coli is regulated by a short RNA complementary to a region of ompF RNA encompassing the "Shine-Dalgarno' sequence and the translation initiation codon . This RNA has been termed micRNA (messenger-RNA-interfering complementary RNA), and its discovery has prompted us to construct an artificial micRNA system designed to regulate gene expression in E . coli . A given target gene can be repressed by artificially producing an RNA (micRNA) complementary to the mRNA encoded by that gene . A micRNA system has also been used successfully in tissue-cultured mammalian cells . The use of artificial micRNAs to specifically regulate individual genes has great potential as a novel cellular immune system for blocking bacteriophage or virus infection . Here, we report that on induction of micRNAs directed against the coat protein and/or the replicase of the E . coli bacteriophage SP, phage proliferation was effectively prevented . We propose that the micRNA immune system provides an effective means of preventing viral infection as well as the expression of harmful genes in both prokaryotes and eukaryotes.

FEBS Lett, 1985 Jun 3, 185(1), 197 - 202
Unusual frequencies of certain alternating purine-pyrimidine runs in natural DNA sequences: relation to Z-DNA; Trifonov EN et al.; Prokaryotic, eukaryotic and mitochondrial DNA sequences of total Length 300 000 nucleotides have been analyzed to find out whether stretches of alternating purines and pyrimidines are unusual in terms of occurrence, composition and base sequence . Alternating runs longer than 5 nucleotides are significantly under-represented in the natural sequences as compared to random ones . Octanucleotides are the most deficient, occurring at only 60% of the frequency expected in random sequences . An unexpectedly high proportion of these octamers consists of alternating tetramers with the repeat structure (PuPyPuPy)2 or (PyPuPyPu)2 . DNA stretches containing such sequences can potentially form a S1 nuclease sensitive slippage (staggered loop) structure, which might serve as a locally unstacked intermediate in the B- to Z-DNA conformational transition.

Nature, 1985 Jun 27-Jul 3, 315(6022), 771 - 3
Myxococcus xanthus spore coat protein S may have a similar structure to vertebrate lens beta gamma-crystallins; Wistow G et al.; The Gram-negative bacterium Myxococcus xanthus has a complex life cycle during which large amounts of a protein of relative molecular mass (Mr) 19,000, known as protein S, are assembled into a spore surface coat by a process that specifically requires calcium ions . The gene for protein S has been cloned and the DNA sequence shows that the gene product is composed of four internally repeated homologous sequences, each 40 amino acids long . Although protein S resembles calmodulin both in its internally duplicated structure and its ability to bind calcium, it apparently has a beta-sheet secondary structure rather than the helix-loop-helix motifs that characterize the calmodulin family . We now show that protein S has a striking homology with the beta- and gamma-crystallins of the vertebrate eye lens which are beta-sheet proteins with internally duplicated structures . This implies that the beta- and gamma-crystallins evolved from already existing proteins, whose ancestors occurred in the prokaryotes . The biological function of protein S, as a closely packed, stable protein in a relatively dehydrated environment, has implications for the functions of crystallins, which are found closely packed in the lens fibre cells, where their stability is essential for maintenance of transparency.

Eur J Biochem, 1985 Jun 3, 149(2), 353 - 61
Do yeast aminoacyl-tRNA synthetases exist as soluble enzymes within the cytoplasm?
Cirakoglu B, Waller JP.
The aminoacyl-tRNA synthetases from a crude extract of yeast were shown to bind to heparin-Ultrogel through ionic interactions, in conditions where the corresponding enzymes from Escherichia coli did not . The behaviour of purified lysyl-tRNA synthetases from yeast and E . coli was examined in detail . The native dimeric enzyme from yeast (Mr 2 X 73000) strongly interacted with immobilized heparin or tRNA, as well as with negatively charged liposomes, in conditions where the corresponding native enzyme from E . coli (Mr 2 X 65000) displayed no affinity for these supports . Moreover, the aptitude of the native enzyme from yeast to interact with polyanionic carriers was lost on proteolytic conversion to a fully active modified dimer of Mr 2 X 65500 . A structural model is proposed, according to which each subunit of yeast lysyl-tRNA synthetase is composed of a functional domain similar in size to that of the prokaryotic enzyme, contiguous to a 'binding' domain responsible for association to negatively charged carriers . The evolutionary acquisition of this property by lower eukaryotic aminoacyl-tRNA synthetases suggests that it fulfils an important function in vivo, unrelated to catalysis . We propose that it promotes the compartmentalization of these enzymes within the cytoplasm, through associations with as yet unidentified, negatively charged components, by electrostatic interactions too fragile to withstand the usual extraction conditions.

Cell, 1985 Jun, 41(2), 375 - 82
Processing of the intron-containing thymidylate synthase (td) gene of phage T4 is at the RNA level; Belfort M et al.; The interrupted T4 phage td gene, which encodes thymidylate synthase, is the first known example of an intron-containing prokaryotic structural gene . Analysis of td-encoded transcripts provides evidence in favor of maturation at the RNA level . Northern blotting with T4 RNA and with region-specific probes revealed three classes of RNA: diffuse premessage (ca . 2.5 kb), a low-abundance mature mRNA (ca . 1.3 kb), and an abundant free intron RNA (ca . 1.0 kb) . The existence of covalently joined mature mRNA was suggested by hybridization and S1 protection experiments and was confirmed by primer extension analysis of the splice junction . In analogy to expression of interrupted eukaryotic genes, these results are consistent with an RNA processing model that would account for the direct gene transcript serving as precursor for both free intron RNA and a spliced mRNA that is colinear with the thymidylate synthase product.

Biosci Rep, 1985 Jun, 5(6), 453 - 61
Functional prokaryotic gene control signals within a eukaryotic rainbow trout protamine promoter; Jankowski JM et al.; Following the construction of a series of pSV2-cat derived plasmids containing the chloramphenicol acetyltransferase (CAT) gene under the control of a eukaryotic trout protamine promoter, it was noted that Escherichia coli, transformed with these plasmids, developed resistance to chloramphenicol (CM) . This result suggested that the eukaryotic trout protamine promoter possessed significant prokaryotic promoter activity . Modification of the trout protamine promoter region by removing the region containing the eukaryotic Goldberg-Hogness box in the plasmid p525-cat increased the expression of the CAT gene almost to the wild-type level and conferred strong CM resistance . Sequence comparisons of the plasmid series indicate that prokaryotic promoter elements are present in the trout protamine promoter and that their similarity to the prokaryotic promoter consensus sequences and the distance between the two elements is more favourable in p525-cat, the plasmid which confers the greatest CM resistance.

J Immunol, 1985 Jun, 134(6), 4189 - 93
In vitro expression of factor-mediated cytotoxic activity generated during the immune response to Chlamydia in the mouse; Byrne GI et al.; Supernatant fluid (SF) prepared by mitogen incubation of spleen cells from A/J mice previously immunized against lethal challenge by the 6BC strain of Chlamydia psittaci was cytotoxic for mouse fibroblasts (L cells) infected with 6BC, as detected by the {3H}thymidine release assay and the trypan blue exclusion test . In contrast, SF prepared from spleen cells taken from unimmunized animals (controls) was not cytotoxic when added to infected L cells . No cytotoxicity was observed when SF was added to uninfected L cells . Maximal levels of cytotoxicity were observed only from cells infected with 6BC for at least 26 hr and exposed to SF for greater than 20 hr . Furthermore, the degree of cytotoxicity was dependent on both the dose of Chlamydia administered and the concentration of SF in the medium . We conclude that the capacity to secrete a spleen cell cytotoxic factor is an aspect of the immune response against the obligate intracellular prokaryotic pathogen Chlamydia . Our results indicate that SF-mediated cytotoxicity is induced subsequent to immunization with Chlamydia, and is significantly more pronounced against infected as opposed to uninfected L cells.

Mutat Res, 1985 Jun-Jul, 150(1-2), 99 - 105
Microinjection of Micrococcus luteus UV-endonuclease restores UV-induced unscheduled DNA synthesis in cells of 9 xeroderma pigmentosum complementation groups; de Jonge AJ et al.; The UV-induced unscheduled DNA synthesis (UDS) in cultured cells of excision-deficient xeroderma pigmentosum (XP) complementation groups A through I was assayed after injection of Micrococcus luteus UV-endonuclease using glass microneedles . In all complementation groups a restoration of the UV-induced UDS, in some cells to the repair-proficient human level, was observed . Another prokaryotic DNA-repair enzyme, T4 endonuclease V, restored the UV-induced UDS in a similar way after microinjection into XP cells . Since both enzymes specifically catalyse only the incision of UV-irradiated DNA, we conclude that this activity is impaired in cells of all 9 excision-deficient XP complementation groups tested.

Biochimie, 1985 Jun, 67(6), 563 - 82
{Mode of action of cyclic amp in prokaryotes and eukaryotes, CAP and cAMP-dependent protein kinases}; de Gunzburg J; cAMP is an ubiquitous compound which is involved in the regulation of many biological processes . In bacteria such as E . coli, cAMP mediates the activation of catabolic operons via the CAP protein . The CAP-cAMP complex, whose tridimensional structure has recently been established, binds to the promoter regions of catabolic operons at a specific site, and activates their transcription by inducing RNA polymerase to bind and initiate transcription at the correct site . Various phenomenons including protein-protein interactions or CAP-induced DNA bending or kinking could be involved in the process of forming the open transcription complex . In eukaryotes, cAMP activates cAMP dependent protein kinases which covalently modify proteins by phosphorylation on serine or threonine residues . The catalytically inactive holoenzyme is generally a tetramer containing two regulatory subunits, each capable of binding two molecules of cAMP, and two catalytic subunits . In mammalian cells, two types of cAMP dependent protein kinases (I and II) can be distinguished on the basis of their regulatory subunits; their relative proportion varies from tissue to tissue . Binding of cAMP to the regulatory subunits induces the dissociation of the holoenzyme and releases the free and active catalytic subunits . Phosphorylation of proteins occurs at sequences containing two basic residues in the vicinity of the phosphorylated serine or threonine . A heat-stable protein, present in most eukaryotic cells, specifically interacts with the catalytic subunit and inhibits its activity . The amino-acid sequence of cAMP dependent protein kinases has recently been determined . It is interesting to note that the domains responsible for cAMP binding by the regulatory subunits of mammalian cAMP dependent protein kinases and CAP share important sequence homologies . The same phenomenon is observed concerning the domain responsible for ATP binding to the catalytic subunit of cAMP dependent protein kinases and that of tyrosine-specific protein kinases from oncoviruses . Other eukaryotic proteins such as S-adenosyl-L-homocysteine (SAH) hydrolase are also capable of binding cAMP . The latter is involved in the regulation of S-adenosyl-L-methionine dependent methylations, and its activity could be affected by cAMP . Besides its role as an effector of enzymatic activity via phosphorylation, such as in the regulation of glycogen metabolism, cAMP has recently been shown to activate the transcription of a number of eukaryotic genes . This process probably also involves protein phosphorylation, but its precise mechanism remains to be understood.

Virus Res, 1985 Jun, 2(4), 291 - 9
Sequence of the serotype-specific glycoprotein of the human rotavirus Wa strain and comparison with other human rotavirus serotypes; Mason BB et al.; Complementary DNA was synthesized from the double-stranded RNA of the Wa strain of human rotavirus and inserted into the bacterial plasmid pBR322 . Clones which contained the gene that codes for the viral glycoprotein (VP7) were identified and the nucleotide sequence was determined . The gene was 1062 base pairs in length with an open reading frame which coded for 326 amino acids . Two potential glycosylation sites were found as well as two hydrophobic regions at the N-terminus of the polypeptide . The untranslated regions at the 5' and 3' ends were 48 base pairs and 33 base pairs long, respectively . Only one nucleotide at position 493 differed from the sequence of the Wa VP7 gene described by Richardson et al . (1984, J . Virol . 51, 860-862) . A strong prokaryotic promoter sequence was also found between residues 434 and 462 . A comparison of the amino acid sequence of the Wa strain (serotype 1) to the Hu/5 strain of human rotavirus (serotype 2) and SA11, the simian rotavirus (serotype 3), revealed a high degree of homology (79.1% and 83.1%, respectively) between the serotypes, suggesting that rotavirus serotypes are stable . The hydrophilic regions of VP7 of the three serotypes were identified and compared for homology . Four of these regions showed variation between serotypes.

J Mol Biol, 1985 May 25, 183(2), 117 - 28
Primary structure of the hip gene of Escherichia coli and of its product, the beta subunit of integration host factor; Flamm EL et al.; We describe the isolation and sequencing of the hip gene of Escherichia coli and show that it encodes the beta subunit of integration host factor (IHF beta) . In order to locate the coding region, we constructed a set of deletion mutants by exonucleolytic digestion of a fragment containing hip, determined which mutants were hip+ and which hip- by complementation, and then sequenced the ends of the critical deletions . The 5' end of the coding region was located precisely by comparing the deduced amino acid sequence to the actual N-terminal amino acid sequence of IHF . Our assignment of the coding region was further substantiated by the nucleotide sequences of a hip point mutant and of internal replacement mutations . We found a probable promoter for hip located about 85 base-pairs upstream from the initial AUG codon and about 75 base-pairs downstream from the 3' end of the neighboring gene, rpsA, and we constructed an IHF beta overproducer by fusing the coding sequences to the lambda pL promoter . A survey of known protein sequences revealed a close relationship between IHF beta and the type II prokaryotic DNA binding proteins (the "histone-like" proteins) . This relationship is shared to a considerable extent by the other subunit of IHF, IHF alpha . A hip missense mutation that replaces a completely conserved glycine with aspartate has a null phenotype, suggesting that the conserved regions are functionally important.

J Biol Chem, 1985 May 25, 260(10), 6160 - 6
Linear diffusion of restriction endonucleases on DNA; Ehbrecht HJ et al.; We have investigated the dependence of the rate of cleavage of DNA by EcoRI, HindIII, and BamHI on the chain length of the substrate . In order to keep the influence of flanking sequences and of nonspecific binding identical for all substrates we have carried out all experiments with the same plasmid DNA which had been digested previously with a variety of different restriction enzymes to give a set of substrates of different lengths . Our results show that depending on the buffer conditions long substrates are cleaved faster than small ones . We interpret these findings to mean that under certain conditions a linear diffusion of the enzymes on the DNA is involved in localizing the recognition sites . For EcoRI the mean diffusion length is approximately 1000 base pairs at 1 mM MgC12 which can be shown by diffusion theory to correspond to a linear diffusion coefficient of 5 X 10(-10) cm2 s-1 . At 10 mM MgCl2 the linear diffusion of EcoRI is negligible and does not lead to a significant enhancement of the rate of site localization . In the presence of nonsaturating amounts of one of the prokaryotic histone-like protein Hu (NS 2) small and large DNA substrate are cleaved with identical rate by EcoRI indicating that other proteins bound to the DNA constitute a barrier across which linear diffusion cannot take place . We conclude that linear diffusion, albeit detectable under certain conditions in vitro, probably is of little importance for the process of site localization in vivo.

J Biol Chem, 1985 May 10, 260(9), 5240 - 3
Expression of nitrogen fixation genes in foreign hosts . Assembly of nitrogenase Fe protein in Escherichia coli and in yeast; Berman J et al.; In Klebsiella pneumoniae, the nifH gene encodes the Fe protein (Kp2) polypeptide that is assembled into a homodimer responsible for the reduction of nitrogenase . Escherichia coli or the yeast Saccharomyces cerevisiae, transformed with the K . pneumoniae nifH gene in suitable expression vectors, synthesize the Fe protein polypeptide . This study examines the assembly of the nifH gene product into its characteristic dimeric structure in E . coli and in yeast . Immunoblotting methods, as well as 55Fe2- labeling of K . pneumoniae were employed to detect native nitrogenase components in cell lysates . E . coli and yeast transformants contained a protein similar to native Kp2 in its immunoreactivity, apparent molecular weight, and lability in the presence of oxygen or MgATP . While in E . coli the co-introduction of nifH and nifM resulted in enhanced levels of the nifH product, it appears that the nifH gene product alone is sufficient for the assembly of an Fe protein-like structure in foreign prokaryotic and eukaryotic hosts.

Science, 1985 May 10, 228(4700), 719 - 22
Coupling of Tetrahymena ribosomal RNA splicing to beta-galactosidase expression in Escherichia coli; Price JV et al.; Splicing of the Tetrahymena ribosomal RNA precursor is mediated by the folded structure of the RNA molecule and therefore occurs in the absence of any protein in vitro . The Tetrahymena intervening sequence (IVS) has been inserted into the gene for the alpha-donor fragment of beta-galactosidase in a recombinant plasmid . Production of functional beta-galactosidase is dependent on RNA splicing in vivo in Escherichia coli . Thus RNA self-splicing can occur at a rate sufficient to support gene expression in a prokaryote, despite the likely presence of ribosomes on the nascent RNA . The beta-galactosidase messenger RNA splicing system provides a useful method for screening for splicing-defective mutations, several of which have been characterized.

Science, 1985 May 10, 228(4700), 742 - 4
Expression of the chlamydial genus-specific lipopolysaccharide epitope in Escherichia coli; Nano FE et al.; The obligate intracellular prokaryote Chlamydia trachomatis is the etiological agent of trachoma and is a primary causative pathogen of sexually transmitted genital tract disease; both diseases affect millions of people each year . The cloning of genes encoding the enzyme or enzymes producing the genus-specific lipopolysaccharide antigen of Chlamydia into Escherichia coli is reported here . The cloned chlamydial lipopolysaccharide antigen appears to be a hybrid lipopolysaccharide molecule composed of both Chlamydia and Escherichia coli components . The chlamydial lipopolysaccharide antigen is expressed on the surfaces of the viable Escherichia coli recombinants . These findings may have a significant impact on defining the role of this highly conserved antigen in the pathogenesis and diagnosis of chlamydial infections.

Proc Natl Acad Sci U S A, 1985 May, 82(9), 2731 - 5
Nucleotide sequence and organization of the mouse adenine phosphoribosyltransferase gene: presence of a coding region common to animal and bacterial phosphoribosyltransferases that has a variable intron/exon arrangement; Dush MK et al.; We have determined the nucleotide sequence of a functional mouse adenine phosphoribosyltransferase (APRT) gene and its cDNA . The amino acid sequence of the enzyme is deduced from an open reading frame in the cDNA and predicts a protein with a molecular weight of 19,560 . The protein coding region of the gene is approximately 2 kilobases, and it is composed of five exons and four introns . While the body of the gene is 53% G + C, the 200 nucleotides upstream from the ATG translation start codon are 66% G + C and contain three copies of the sequence C-C-G-C-C-C . The mouse APRT enzyme shares a homologous 20-amino acid sequence with mouse, hamster, and human hypoxanthine phosphoribosyltransferases (HPRTs) and several bacterial phosphoribosyltransferases . This sequence has previously been shown to be a likely catalytic domain in human HPRT and Escherichia coli glutamine phosphoribosyltransferase . Because of the similarities in function of these proteins, both eukaryotic and prokaryotic, it is not unexpected that they should exhibit one or more regions of homology, particularly at the 5-phosphoribosyl-1-pyrophosphate and purine binding sites, especially if they are related via a common evolutionary lineage . This homologous sequence is interrupted by a single intron in the mouse APRT gene and by two introns in the mouse HPRT gene . Furthermore, the positions of both introns in the HPRT sequence are different from that of the single intron in the corresponding sequence of the APRT gene.

Biochimie, 1985 May, 67(5), 549 - 53
Search for promoter sites of prokaryotic DNA using learning techniques; Sallantin J et al.; Using learning techniques previously described in this journal, we have built an expert system able to point to the start DNA point of a sequence and therefore to recognize a promoter . However, to build this system, we have focused on the TATA box and its environment . We have used this expert system to look for new promoters and also to construct new promoters . The results obtained are discussed.

EMBO J, 1985 May, 4(5), 1301 - 6
The promoter of the late p10 gene in the insect nuclear polyhedrosis virus Autographa californica: activation by viral gene products and sensitivity to DNA methylation; Knebel D et al.; In lepidopteran insect cells infected with the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV), two major late viral gene products are expressed: the polyhedrin, a 28 000 mol . wt . protein which makes up the mass of the nuclear inclusion bodies, and a 10 000 mol . wt . protein (p10) whose function is unknown . The nucleotide sequences of these strong promoters conform to those of other eukaryotic promoters and are rich in AT base pairs . We used the pSVO-CAT construct containing the prokaryotic gene chloramphenicol acetyl transferase (CAT) to study the function of the p10 gene promoter in insect and mammalian cells . Upon transfection of the pAcp10-CAT construct, which contained 402 bp of the p10 gene of AcNPV DNA in the HindIII site of pSVO-CAT, CAT activity was determined . The p10 gene promoter was inactive in human HeLa cells and in uninfected Spodoptera frugiperda insect cells . The same promoter was active, however, in AcNPV-infected S . frugiperda cells and exhibited optimal activity when cells were transfected 18 h after infection with the insect virus . This finding demonstrated directly that the p10 gene promoter required other viral gene products for its activity in insect cells . The nature of these products was unknown . The p10 gene promoter sequence contained one 5'-CCGG-3' site 40 bp upstream from the cap site of the gene and two such sites 178 and 192 bp downstream from the ATG initiation codon of the gene . Since Drosophila DNA or S . frugiperda DNA contained no 5-methylcytosine or extremely small amounts of it, we were interested in determining the effect of site-specific methylations on the p10 gene insect virus promoter . Methylation at the 5'-CCGG-3' sites led to a block of this promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Proc Natl Acad Sci U S A, 1985 May, 82(9), 2674 - 8
Growth-dependent expression of multiple species of DNA methyltransferase in murine erythroleukemia cells; Bestor TH et al.; Friend murine erythroleukemia cells were found to contain three distinct species of DNA (cytosine-5-)-methyltransferase (DNA MeTase) whose relative proportions were a characteristic function of the proliferative state of the cells . Rapidly proliferating cells contained a Mr 190,000 species of DNA MeTase (DNA MeTase III), whereas cells in the late logarithmic/early plateau phase of cellular growth contained two species of Mr 150,000 and 175,000 (DNA MeTase I and II); stationary phase cells contained primarily DNA MeTase I . The three species of DNA MeTase displayed structural similarities, as determined by analysis of partial proteolysis products, and have similar de novo sequence specificities in transmethylation reactions involving purified enzyme and prokaryotic DNA . The different relative proportions of the enzymes in cells under different growth conditions suggest that the three species of DNA MeTase fulfill different roles in processes leading to the perpetuation of DNA methylation patterns.

Proc Natl Acad Sci U S A, 1985 May, 82(9), 2652 - 6
The 140-kDa adenovirus DNA polymerase is recognized by antibodies to Escherichia coli-synthesized determinants predicted from an open reading frame on the adenovirus genome; Friefeld BR et al.; Sequence studies of the adenovirus 2 genome have revealed the presence of a large open reading frame (ORF) from 22.9 to 14.2 map units that is believed to encode most of the adenovirus DNA polymerase (Ad Pol) . An 838-base-pair fragment (19.6-17.3 map units) containing approximately 25% of this ORF has been cloned and expressed in a beta-galactosidase-chloramphenicol acetyltransferase (lacZ-CAT) expression vector under the control of the trp-lac hybrid promoter . This recombinant vector directed the synthesis of a 58-kDa lacZ-Ad Pol-CAT fusion protein that has CAT activity . This fusion protein was easily purified by affinity chromatography in which chloramphenicol, the substrate for CAT, was covalently bound to a matrix . Antisera were prepared against the purified 58-kDa lacZ-Ad Pol-CAT fusion protein and were found to react specifically with the 140-kDa Ad Pol by ELISA and immunoblot analysis . In addition, these antisera recognized 120- and 29-kDa polypeptides in immunoblot analysis of partially purified terminal protein precursor (pTP)-Ad Pol complex . The exact nature of the 120- and 29-kDa polypeptides is not known, but they may be breakdown products of Ad Pol . Although the lacZ-Ad Pol-CAT fusion protein is not active in any of the Ad Pol enzymatic reactions, antibody against the prokaryotic fusion protein should be useful for screening bacteria harboring plasmids that have been constructed to express the entire Ad Pol ORF.

Biochimie, 1985 May, 67(5), 533 - 9
Localization of the initiation of translation in messenger RNAs of prokaryotes by learning techniques; Rodier F et al.; Learning processes are applied to the recognition of protein coding regions in prokaryotes . Non-contradictory, statistical and logical rules are deduced from a set of known examples of coding sequences . These rules enable to build characteristic patterns on the m-RNA upstream of the initiating codon . These rules are applied with success to recognize more than 180 coding sequences and to detect and/or eliminate hypothetical reading frames or unknown genes.

Nucleic Acids Res, 1985 Apr 11, 13(7), 2281 - 92
The wheat mitochondrial gene for apocytochrome b: absence of a prokaryotic ribosome binding site; Boer PH et al.; The wheat mitochondrial gene for apocytochrome b (CYB) has been identified by its hybridization to a yeast CYB probe and its nucleotide sequence has been determined . The wheat CYB sequence predicts a cytochrome b apoprotein of 398 amino acids; it is almost identical to that of maize but has ten additional amino acids at the carboxy terminus . No introns are present in the wheat CYB gene, but an internal segment of the gene is repeated at another genomic location . Transcript analysis reveals a single wheat CYB mRNA of approximately 2.4 kb with a long untranslated leader . Sequences upstream of the CYB coding region are very similar in wheat and maize but the stretch proposed to be a ribosome binding site in maize is not conserved in wheat . The corresponding leader regions of the wheat mitochondrial mRNAs for cytochrome oxidase subunits I and II also lack complementarity to the 3'-end of the small subunit rRNA . We conclude that alternative signals are involved in the initiation of translation in plant mitochondria.

Nature, 1985 Apr 11-17, 314(6011), 556 - 8
ATP-dependent specific binding of Tn3 transposase to Tn3 inverted repeats; Wishart WL et al.; Transposons are discrete segments of DNA which are capable of moving from one site in a genome to many different sites . Tn3 is a prokaryotic transposon which is 4,957 base pairs (bp) long and encodes a transposase protein which is essential for transposition . We report here a simple method for purifying Tn3 transposase and demonstrate that the transposase protein binds specifically to the ends of the Tn3 transposon in an ATP-dependent manner . The transposase protein binds to linear double-stranded DNA both nonspecifically and specifically; the nonspecific DNA binding activity is sensitive to challenge with heparin . Site-specific DNA binding to the ends (inverted repeats) of Tn3 is observed only when binding is performed in the presence of ATP; this ATP-dependent site-specific DNA binding activity is resistant to heparin challenge . Our results indicate that ATP qualitatively alters the DNA binding activity of the transposase protein so that the protein is able to bind specifically to the ends of the Tn3 transposon.

Proc Natl Acad Sci U S A, 1985 Apr, 82(8), 2306 - 9
UGA is read as tryptophan in Mycoplasma capricolum; Yamao F et al.; UGA is a nonsense or termination (opal) codon throughout prokaryotes and eukaryotes . However, mitochondria use not only UGG but also UGA as a tryptophan codon . Here, we show that UGA also codes for tryptophan in Mycoplasma capricolum, a wall-less bacterium having a genome only 20-25% the size of the Escherichia coli genome . This conclusion is based on the following evidence . First, the nucleotide sequence of the S3 and L16 ribosomal protein genes from M . capricolum includes UGA codons in the reading frames; they appear at positions corresponding to tryptophan in E . coli S3 and L16 . Second, a tRNATrp gene and its product tRNA found in M . capricolum have the anticodon sequence 5' U-C-A 3', which can form a complementary base-pairing interaction with UGA.

Cell, 1985 Apr, 40(4), 933 - 41
Novel partitioning of DNA cleavage sites for Drosophila topoisomerase II; Udvardy A et al.; We have examined the long-range distribution of double-stranded DNA cleavage sites for Drosophila melanogaster topoisomerase II . These studies reveal a novel partitioning of preferred topoisomerase II cleavage sites . In the eukaryotic DNAs examined, major cleavage sites were typically found in nontranscribed spacer segments and close to the 5' and 3' boundaries of genes . In contrast, there were few if any prominent cleavage sites within genes . In addition, most of the major topoisomerase II cleavage sites closely corresponded to naked DNA hypersensitive sites for the prokaryotic enzyme, micrococcal nuclease.

FEBS Lett, 1985 Mar 25, 182(2), 470 - 4
Identification of lipoglycan antigens from the Acholeplasma laidlawii cell membrane in crossed immunoelectrophoresis; Johansson KE et al.; Membranes from the wall-less prokaryote Acholeplasma laidlawii contain a component termed lipoglycan or lipopolysaccharide (LPS) . The lipoglycan has extraction properties, which are similar to those of LPS of gram-negative bacteria, but it is chemically distinct from bacterial LPS . The membrane-bound lipoglycan of A . laidlawii did not seem to be particularly immunogenic and antibodies against it could not always be detected by rocket immunoelectrophoresis (RIE) or crossed immunoelectrophoresis (CIE) in hyperimmune sera raised against membranes . The immunoprecipitate corresponding to the lipoglycan, obtained by CIE of Tween 20-solubilized A . laidlawii membranes, has been identified and shown to be both a cathodically and anodically migrating component at pH 8.6 . The shape of the immunoprecipitate in both RIE and CIE showed that the lipoglycan antigen is composed of at least two components, which are immunologically related.

J Biol Chem, 1985 Mar 25, 260(6), 3852 - 9
Structural relatedness of three ion-transport adenosine triphosphatases around their active sites of phosphorylation; Walderhaug MO et al.; Three membrane-bound adenosine triphosphatases were investigated for homology in the sequence of four amino acids about the active site of phosphorylation . The ATPases were as follows: sodium-potassium-dependent ATPase from dog kidney, Na,K-ATPase; hydrogen-potassium-dependent ATPase from hog gastric mucosa, H,K-ATPase, an ATPase similar to Na,K-ATPase; and an ATPase activity in the plasma membrane of corn, Zea mays, roots (CR-ATPase), a higher plant ATPase . A membrane preparation containing an ATPase of Acholeplasma laidlawii, a prokaryote, (AL) was also investigated . For most of the experiments, the preparations were phosphorylated from {gamma-32P}ATP, denatured in acid, and subjected to proteolytic digestion . Radioactive phosphopeptides were separated by high voltage paper electrophoresis and characterized by sensitivity to chemical reagents . In gastric H,K-ATPase, the aspartate residue at the active site was determined directly by labeling with {3H}borohydride . A common sequence around the active site was found for Na,K-ATPase, H,K-ATPase, and CR-ATPase . This sequence, -Cys-(Ser/Thr)-Asp(P)-Lys-, is similar to that in the calcium ion-transport ATPase of sarcoplasmic reticulum . The AL membrane preparation showed an acylphosphate that turned over rapidly after a chase of labeled membranes with unlabeled ATP . The corresponding sequence was different from that of the three ATPases . An acylphosphate was on two polypeptides with molecular weights of about 80,000 and 60,000; these appear not to correspond to subunits of a Na+-stimulated ATPase in this organism (Lewis, R . N . A . H., and McElhaney, R . N . (1983) Biochim . Biophys . Acta 735, 113-122).

Nucleic Acids Res, 1985 Mar 25, 13(6), 2127 - 39
Complete sequence of IS3; Timmerman KP et al.; We have determined the nucleotide sequence of IS3 . Our IS3 isolate has 39 bp inverted repeats (IR's) with 6 mismatches, and is 1258 bp long . IS3 contains a large open reading frame (ORF) of 288 codons with a smaller, partially overlapping ORF of 91 codons on the opposite strand in codon-codon register . The large ORF is preceded by and has a 4 bp overlap with a 99 codon ORF that has potential transcriptional and translational start signals . Thus, IS3 could encode a bicistronic mRNA . The Shine-Dalgarno sequence for this 99 codon ORF could be sequestered in a stem-loop structure, but only if the transcript began outside IS3, as was first seen with IS10 . This could be a means for preventing fortuitous activation of IS3 by outside promoters . No DNA sequence homology was found between IS3 and other prokaryotic IS elements, but there is slight amino acid sequence homology and significant conservation of hydropathicity patterns between the putative transposases of IS3 and IS2.

Eur J Biochem, 1985 Mar 15, 147(3), 561 - 8
Prokaryotic triterpenoids . The hopanoids of the purple non-sulphur bacterium Rhodomicrobium vannielii: an aminotriol and its aminoacyl derivatives, N-tryptophanyl and N-ornithinyl aminotriol; Neunlist S et al.; Triterpenoids belonging to the hopane family are widely distributed in prokaryotes . Three new hopanoids have now been isolated from the purple non-sulphur bacterium Rhodomicrobium vannielii and identified essentially by spectroscopic methods . The basic compound is the 35-aminobacteriohopane-32,33,34-triol, from which the other two hopanoids are derived by introduction of a tryptophanyl or an ornithinyl moiety linked to the amino group at C-35 via an amide linkage . This is the first report of hopanoids possessing an amino group in their side-chain and linked to aminoacyl residues.

Nucleic Acids Res, 1985 Mar 11, 13(5), 1703 - 16
Characterization of a mitochondrial protein binding to single-stranded DNA; Mignotte B et al.; A DNA-binding protein from Xenopus laevis oocyte mitochondria which has been found associated with the D-loop also shows a strong preference for single-stranded DNA . The binding to polynucleotides is dependent on the base composition, but no sequence specificity was found . This protein, called mtSSB, binds tightly and cooperatively to single-stranded DNA . By its amino-acid composition and its binding properties it appears to be similar to the single-stranded DNA-binding proteins found in prokaryotes.

FEBS Lett, 1985 Mar 11, 182(1), 163 - 6
Citrate synthase: an immunochemical investigation of interspecies diversity; Pullen AM et al.; Rabbit antibodies have been raised to pig heart citrate synthase . Using purified IgG, competitive enzyme-linked immunoassays and assays of citrate synthase activity indicate the presence of antibodies to a number of antigenic sites on the enzyme, only some of which are essential for catalytic activity . From a comparison of citrate synthases from prokaryotic and eukaryotic organisms, the degree of interaction between antibody and enzyme was in the order: pig heart greater than pigeon breast greater than Bacillus megaterium greater than Escherichia coli . These findings are discussed in terms of the known interspecies diversity of the enzyme.

J Biol Chem, 1985 Mar 10, 260(5), 3126 - 31
Euglena gracilis chloroplast elongation factor Tu . Purification and initial characterization; Sreedharan SP et al.; The chloroplast protein synthesis elongation factor Tu (EF-Tuchl) has been purified to near homogeneity from Euglena gracilis . Chromatography of the postribosomal supernatant of light-induced Euglena on DEAE-Sephadex reveals two forms of EF-Tuchl . Further purification has shown that one species consists of a complex between EF-Tuchl and a factor that stimulates its activity . The other species consists of free EF-TUchl . The factor has been purified from both chromatographic forms by taking advantage of the molecular weight shift that occurs upon disruption of the complex between EF-Tuchl and the stimulatory factor . EF-Tuchl consists of a single polypeptide chain with a molecular weight of about 50,000 . EF-Tuchl is as active on Escherichia coli ribosomes as it is on its homologous ribosomes but displays no detectable activity on eukaryotic cytoplasmic ribosomes . It is stimulated in polymerization by E . coli EF-Ts and will form a complex with the prokaryotic factor that can be isolated by gel filtration chromatography . Like E . coli EF-Tu, it is sensitive to modification by N-ethylmaleimide and is inhibited by the antibiotic kirromycin . Thus, the chloroplast factor has many features that reflect the close relationship between prokaryotic and chloroplast translational systems.

Biochim Biophys Acta, 1985 Mar 8, 838(3), 312 - 20
The circular dichroism properties of phi W-14 DNA containing alpha-putrescinylthymine; Spetter S et al.; The circular dichroism properties of phi W-14 DNA containing alpha-putrescinylthymine and its acetylated derivative have been examined in a number of aqueous solvents . Native phi W-14 DNA exhibits a B-type CD spectrum whose characteristics do not entirely conform to what would be expected for its GC content (51%) . The conformationally sensitive positive band above 260 nm has a rotational strength greater than that normally found in prokaryotic DNAs of comparable GC content, such as Escherichia coli DNA . The rotational strength of this band in the spectrum of the heat-denatured form of phi W-14 DNA, however, is similar to that of heat denatured E . coli DNA . Abolition of the positive charge on the putrescine residues of native phi W-14 DNA by reaction with CH2O or by acetylation reduces the rotational strength to a level appropriate for its GC content . Increases in the electrolyte content of the solvent have the same effect, although the rotational strength of this band in phi W-14 DNA does not become comparable to that of E . coli DNA until 6-7 M LiCl . Titration to pH 10.6 in solvents of modest electrolyte content, however, fails to appreciably affect the CD spectral properties of either native phi W-14 DNA or the derivative in which half of the secondary and all of the primary amino groups have been acetylated . On the basis of these results we have concluded that the enhanced rotational strength of the positive band above 260 nm in the CD spectrum of phi W-14 DNA is due to a conformational difference caused by an ion-pair interaction of the positively charged primary amino groups of putrescine with the phosphate backbone . The CD spectral properties, however, reveal that these differences, averaged over the entire basepair population, appear to be relatively small . The average conformation, at least in dilute aqueous solutions, seems to be an unexceptional B variant with conformational properties which would be more appropriate for a DNA of higher CG content.

J Mol Biol, 1985 Mar 5, 182(1), 131 - 49
Structure and polymorphism of bipolar isopranyl ether lipids from archaebacteria; Gulik A et al.; We describe in this work the structure and polymorphism of a variety of lipids extracted from Sulfolobus solfataricus, an extreme thermoacidophilic archaebacterium growing at about 85 degrees C and pH 2 . These lipids are quite different from the usual fatty acid lipids of eukaryotes and prokaryotes: each molecule consists of two C40 omega-omega' biphytanyl residues (with 0 to 4 cyclopentane groups per residue), ether linked at both ends to two (variably substituted) glycerol or nonitol groups . Four lipid preparations were studied; the total and the polar lipid extracts, and two hydrolytic fractions, the symmetric glycerol dialkyl glycerol tetraether and the asymmetric glycerol dialkyl nonitol tetraether, as a function of water content and temperature, using X-ray scattering techniques . The main conclusions from the study of the four lipid preparations can be summarized as follows . (1) As with other lipids, a remarkable number and variety of phases are observed over a temperature-concentration range close to "physiological" conditions . The possibility is discussed that this polymorphism reflects a fundamental property of lipids, closely related to their physiological role . (2) As in other lipids, two types of chain conformations are observed: a disordered one (type alpha) at high temperature; at lower temperature, a more ordered packing of stiff chains, all parallel to each other (type beta') . At temperatures and degrees of hydration approaching the conditions prevailing in the living cell, the conformation is of type alpha . (3) In all the phases with chains in the alpha conformation, the unsubstituted glycerol headgroups, whose concentration is high in these lipids, segregate in the hydrocarbon matrix, away from the other polar groups . This property may have interesting biological consequences: for example, the chains of a fraction of the bipolar lipid molecules can span hydrocarbon gaps as wide as 75 A . (4) Two cubic phases are observed in the total and the polar lipid extracts, which display a remarkable degree of metastability, most unusual in lipid phase transitions involving structures with chains in the alpha conformation . This phenomenon can be explained by the interplay of the physical structure of the cubic phases (the two contain two intertwined and unconnected three-dimensional networks of rods) and the chemical structure of the lipid molecules: the two headgroups of most molecules being anchored on each of the two networks of rods, the migration of the lipid molecules is hindered by the two independent diffusion processes and by the entanglement of the chains.(ABSTRACT TRUNCATED AT 400 WORDS)

J Gen Microbiol, 1985 Mar, 131 ( Pt 3), 581 - 90
Isolation and characterization of small heat-stable acid-soluble DNA-binding proteins from Bacillus subtilis nucleoids; Salti V et al.; Small heat-stable, acid-soluble proteins (HASP) have been isolated from Bacillus subtilis nucleoids obtained from cell lysates of low ionic strength and lysozyme concentration . They were identified by their ability to bind homologous and heterologous native and denatured DNA . Four major species, of 8.5, 12, 23 and 26 kDal, were found . Their affinity for DNA was moderate as measured by the sensitivity to ionic strength of the DNA-protein complex (0.1-0.4 M-NaCl) . Partial digestion by micrococcal nuclease of the 'low ionic strength nucleoids' released a DNA fragment of 80-120 bp . The data reported here indicate that small basic proteins, together with other components such as RNA, cations and polyamines, may be involved in the compaction of the prokaryotic genome.

Biokhimiia, 1985 Mar, 50(3), 459 - 64
{Comparison of eukaryotic and prokaryotic elongation factors: isoelectric points and molecular masses.}; Minich WB et al.; Using the O'Farrell method, a two-dimensional analysis of RNA-binding proteins from rabbit reticulocytes was carried out . The latter have been shown to consist of several scores of polypeptides, predominantly of a moderately basic type with isoelectric points ranging from 7 to 9.5 . The two main components of RNA-binding proteins have been identified as eukaryotic elongation factors EF-1L and EF-2 . The RNA-binding elongation factors in eukaryotes have higher isoelectric points and somewhat higher molecular masses as compared to their functional analogs from prokaryotes EF-Tu and EF-G having no affinity for RNA . These results are compatible with the assumption that a nonspecific RNA-binding ability of elongation factors in eukaryotes could have arisen in the course of evolution due to the appearance of an additional RNA-binding "domain" of an alkaline type.

Can J Biochem Cell Biol, 1985 Mar, 63(3), 176 - 82
The nucleotide sequence of tRNAVal3a and tRNAVal3b from Drosophila melanogaster; Addison WR et al.; The nucleotide sequences of two valine tRNA's of Drosophila melanogaster are the following: tRNAVal3a, (sequence in text) tRNAVal3b, (sequence in text) tRNAVal3a shows greater similarity to prokaryotic and eukaryotic organelle tRNAVal's than does tRNAVal3b.

Proc Natl Acad Sci U S A, 1985 Mar, 82(6), 1628 - 32
Complete sequence of the chicken glyceraldehyde-3-phosphate dehydrogenase gene; Stone EM et al.; Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an evolutionarily conserved glycolytic enzyme, is constitutively expressed in most cell types yet is induced to high levels during the development of fast twitch muscle fibers . To analyze the organization and regulation of the chicken GAPDH gene, we first constructed a nearly full-length GAPDH cDNA clone (pGAD-28) . pGAD-28 was used in the current study to screen a genomic library, and several overlapping clones were selected . The GAPDH coding region was detected within a 4.65-kilobase Xho I/EcoRI genomic fragment that was completely sequenced by using the M13 cloning vector system . A small portion of pGAD-28 was used as a primer to extend a 33-nucleotide sequence from the 5' end of GAPDH mRNA . The canonical promoter "TATA" region was found 22 base pairs from the 5' end of the mRNA . The 5' end of the GAPDH gene is extraordinarily G+C-rich (80%) and contains two inverted sequences with a 9-base-pair homology found at -58 (G-G-G-G-C-G-G-G-C) and -93 (G-C-C-C-G-C-C-C-C) nucleotides from the transcription start site . Sequencing also revealed the location of 11 introns within the transcribed portion of the GAPDH gene . The placement of at least 3 of the introns corresponds to the boundaries of protein domains within prokaryotic and eukaryotic GAPDHs that were previously detected by x-ray crystallography . This concordance suggests that introns may have participated in the construction of the earliest GAPDH gene.

Proc Natl Acad Sci U S A, 1985 Mar, 82(5), 1354 - 8
Regulated expression of endonuclease EcoRI in Saccharomyces cerevisiae: nuclear entry and biological consequences; Barnes G et al.; In an investigation to determine how proteins are localized within the nucleus of a cell, we demonstrate that the restriction endonuclease EcoRI is able to enter and function within the nucleus of Saccharomyces cerevisiae when this prokaryotic protein is synthesized in vivo . The EcoRI endonuclease was produced in yeast under the transcriptional control of a regulated yeast promoter by ligating a DNA fragment containing only coding sequences for the endonuclease to the promoter element of the yeast GAL1 gene (the structural gene for galactokinase, EC 2.7.1.6) . Yeast cells harboring a plasmid containing this promoter-gene fusion are able to grow under conditions that repress transcription from the GAL1 promoter . However, under inducing conditions, these yeast cells are unable to grow . Moreover, rad52 mutants, which are deficient in the repair of double-strand breaks, are more sensitive to the presence of the promoter-gene fusion plasmid than are wild-type cells . We demonstrate that the EcoRI endonuclease activity is present in lysates prepared from yeast transformants grown under conditions that induce transcription of GAL1, but this activity is not detectable in cells grown under conditions that repress transcription from the promoter . Furthermore, analysis of yeast chromosomal DNA shows that the endonuclease enters the yeast nucleus and cleaves DNA specifically at EcoRI recognition sites.

J Immunol, 1985 Mar, 134(3), 1888 - 95
Oxidant membrane injury by the neutrophil myeloperoxidase system . I . Characterization of a liposome model and injury by myeloperoxidase, hydrogen peroxide, and halides; Sepe SM et al.; Neutrophils and other phagocytes can injure cells by means of oxygen-dependent mechanisms, particularly the myeloperoxidase (MPO)-H2O2-halide system . The extent of such damage depends in part on the antioxidant defenses of the target cell . To facilitate the study of this phenomenon, we developed a model system in which we employed liposomes as targets for the myeloperoxidase system . The most useful species of liposomes employed 51Cr as the aqueous space marker and phosphatidyl choline with or without dicetyl phosphate and cholesterol as the structural lipid . Marker entrapment was established on the basis of 1) resolution of free from lipid-associated 51Cr by gel exclusion chromatography, 2) latency of 51Cr on rechromatography of detergent-treated liposomes, and 3) a correlation between entrapment and surface charge density . Exposure of liposomes to the complete MPO system resulted in release of 50 to 75% of the entrapped 51Cr . Release was abrogated by omission of myeloperoxidase or H2O2, heating of MPO, or addition of azide, cyanide, or catalase . Reagent H2O2 could be replaced by glucose plus glucose oxidase . Kinetic studies indicated a rapid process, lysis reaching half-maximal levels in less than 2 min . The addition of cyanide at various times interrupted lysis at once, indicating a requirement for ongoing myeloperoxidase-dependent reactions . Liposome disruption by the MPO system was pH dependent, increasing dramatically as pH was decreased from neutrality to 6.0 . In the absence of halides, no lysis was observed . Maximum lysis was found with chloride at 10 to 100 mM, although at 1 mM concentrations, iodide, bromide, and thiocyanate were more active than chloride . Fluoride was inactive . Antagonism between halide species was demonstrated in that low concentrations of iodide or bromide inhibited the effect of optimal concentrations of chloride . Using 125I, we found that exposure of liposomes to the MPO system resulted in an association between iodide and liposomes; moreover, there was a close correspondence between this phenomenon and 51Cr release, suggesting that halogenation may be one mechanism of injury . These studies establish the usefulness of the liposome as a model of oxidant injury by a physiologically relevant system . They bear a striking parallel to work being done on MPO-mediated injury to eukaryotic and prokaryotic cells . By using this simplified model system, it should be possible to explore a number of determinants of target cell injury at a biochemical and molecular level.

Mol Biol Evol, 1985 Mar, 2(2), 92 - 108
Biochemical pathways in prokaryotes can be traced backward through evolutionary time; Jensen RA; For the first time, a credible prokaryotic phylogenetic tree is being assembled by Woese and others using quantitative sequence analysis of oligonucleotides in the highly conservative rRNA . This provides an evolutionary scale against which the evolutionary steps that led to the arrangement and regulation of contemporary biochemical pathways can be measured . This paper presents an emerging evolutionary picture of aromatic amino acid biosynthesis within a large superfamily assemblage of prokaryotes that is sufficiently developed to illustrate a new perspective that will be applicable to many other biochemical pathways.

Biochim Biophys Acta, 1985 Mar 1, 827(3), 439 - 46
Primary structure of peptides from bovine brain glutamine synthetase . Comparison with sequences of glutamine synthetases from other organisms; Johnson RJ et al.; An analysis of the covalent structure of bovine brain glutamine synthetase has been initiated . Cyanogen bromide and tryptic digests have yielded peptides accounting for most of the polypeptide subunit, and sequence analysis has placed in order over half of the amino acids within these peptides . The amino terminus is acetylated and has the following partial sequence: Ac(H, S3, A2, T)-L-B-K-G-I-K-Z-V-Y-M . The carboxyl-terminal sequence is: A-L-P-Q-G-D-K-V-Q-A-M . The peptides isolated from bovine glutamine synthetase show a high degree of homology with peptides isolated from ovine and porcine brain glutamine synthetases . In contrast to the sequence homologies of the proteins from eukaryotic sources, there are no obvious amino acid sequence homologies between bovine brain glutamine synthetase and any prokaryotic glutamine synthetase . Bovine brain glutamine synthetase is inactivated by phenylglyoxal and N-ethylmaleimide . In both cases catalytic activity is protected by the presence of ATP, suggesting the presence of arginine and cysteine residues at or near the ATP binding site.

Am J Hum Genet, 1985 Mar, 37(2), 295 - 310
Transfer of human and murine globin-gene sequences into transgenic mice; Humphries RK et al.; We have studied the transfer of human and murine globin gene sequences into fertilized mouse oocytes by microinjection . Germline transmission was demonstrated for the human delta- and beta-globin genes contained in the bacteriophage lambda H beta G1 . Expression of these human globin-gene sequences was not detectable in either erythroid or nonerythroid tissues . A recombinant plasmid containing the murine beta maj promoter region coupled to the prokaryotic coding sequence for galactokinase was also successfully transferred to two mice, and stable germline transmission of integrated DNA was demonstrated for at least 3 generations . Despite the presence of a murine globin-promoter sequence, expression of the mouse beta maj galactokinase fusion gene was not observed in primary or secondary animals in erythroid or nonerythroid tissues . Analysis of primary and secondary animals from both series of injections revealed extensive de novo methylation in the integrated microinjected DNA . Administration of 5-azacytidine to mice containing the mouse beta maj-promoted galactokinase gene resulted in partial hypomethylation was associated with an apparent two- to threefold increase in galactokinase (gal K) gene expression.

J Biol Chem, 1985 Feb 25, 260(4), 2038 - 41
Bacillus subtilis sigma 28 and Escherichia coli sigma 32 (htpR) are minor sigma factors that display an overlapping promoter specificity; Briat JF et al.; Bacillus subtilis sigma 28-specific promoters (P28) are utilized by a minor form of B . subtilis RNA polymerase (sigma 28RNA polymerase) and not by the predominant RNA polymerases of B . subtilis (sigma 43 RNA polymerase) or Escherichia coli (sigma 70 RNA polymerase) . However B . subtilis P28 are effective promoters in E . coli . This transcription depends on the E . coli htpR+ gene . Similarly, the E . coli rpoD heat shock promoter which is under control of htpR is used in vitro by B . subtilis sigma 28 RNA polymerase . These observations are explained by the fact that E . coli htpR is a minor sigma factor (sigma 32) which shares an overlapping promoter specificity with B . subtilis sigma 28 RNA polymerase . Hence control of bacterial regulons by minor sigma factors is not restricted to Bacilli, or bacteria that carry out a complex differentiation process, but is probably a general, regulatory mechanism in prokaryotes . Transcription from B . subtilis P28 in E . coli does not depend on a heat shock . This suggests that the sequences that control E . coli sigma 32 action are separate from those that control the heat shock regulon . Hence the action of polymerases controlled by minor sigma factors in both B . subtilis and E . coli appears to be controlled by a separate set of regulatory factors.

Nature, 1985 Feb 7-13, 313(6002), 498 - 500
Intron-dependent evolution of chicken glyceraldehyde phosphate dehydrogenase gene; Stone EM et al.; The function of introns in the evolution of genes can be explained in at least two ways: either introns appeared late in evolution and therefore could not have participated in the construction of primordial genes, or RNA splicing and introns existed in the earliest organisms but were lost during the evolution of the modern prokaryotes . The latter alternative allows the possibility of intron participation in the formation of primordial genes before the divergence of modern prokaryotes and eukaryotes . Blake suggested that evidence for intron-facilitated evolution of a gene might be found by comparing the borders of functional protein domains with the placement of introns . We therefore examined glyceraldehyde phosphate dehydrogenase (GAPDH), a glycolytic enzyme, because it is the first protein for which the following data are available: X-ray crystallographic studies demonstrating structurally independent protein 'domains' which were highly conserved during the divergence of prokaryotes and eukaryotes; and a study of genomic organization which mapped introns in the gene . Sequencing of the chicken GAPDH gene revealed 11 introns . We report here that sites of three of the introns (IV, VI and XI) correspond closely with the borders of the NAD-binding, catalytic and helical tail domains of the enzyme, supporting the hypothesis that introns did have a role in the evolution of primitive genes . In addition, other biochemical and structural data were used to construct a model of the intron-mediated assembly of the GAPDH gene that explains the existence of 10 introns.

Nature, 1985 Feb 7-13, 313(6002), 500 - 2
Evolution of catalytic and regulatory sites in phosphorylases; Palm D et al.; Glycogen phosphorylase (E.C.2.4.1.1) was the first enzyme shown to be regulated by allosteric effectors and by protein phosphorylation . Transcriptional control of bacterial phosphorylases further extends the range of regulatory mechanisms by which phosphorylases contribute to the control of carbohydrate metabolism . Despite their regulatory differences, all known phosphorylases share catalytic and structural properties and a strongly conserved pyridoxal-5'-phosphate binding site; this makes phosphorylases highly attractive for investigations into the evolution of regulatory mechanisms . The primary and tertiary structure of rabbit muscle phosphorylase has been determined completely . Recently, comparable amino acid sequences from plants and bacteria have been resolved . Here we report the sequence of 687 amino acids of Escherichia coli maltodextrin phosphorylase, deduced from a cloned malP gene sequence . Alignment of animal and bacterial phosphorylase sequences shows strong homology (48%) throughout 91% of the polypeptide chain enclosing the extrinsic catalytic region . Within this region, structural homology identifies a presumed phosphate-binding site from which the allosteric 5' AMP binding site of rabbit muscle phosphorylase might have developed . From the decreased alignment at the N-terminus and the presence of additional residues compared with bacterial phosphorylases, we conclude that the regulatory sequences that also carry the phosphorylation site in the muscle enzyme were joined to a presumed ancestral precursor gene by gene fusion after separation of the eukaryotic and prokaryotic lines of descent.

Dev Biol, 1985 Feb, 107(2), 281 - 9
Ribosomal-protein synthesis is not autogenously regulated at the translational level in Xenopus laevis; Pierandrei-Amaldi P et al.; Whether ribosomal-protein synthesis in Xenopus laevis is autogenously controlled at the translational level as is known to occur in prokaryotes has been studied . For this purpose ribosomal (r) proteins were prepared from X . laevis ribosomal subunits and group fractionated by ion-exchange chromatography . They were then added to an in vitro translation system directed by an oocyte mRNA fraction which contains template activity for r proteins . The synthesized radioactive products were analyzed by 2D gel electrophoresis and compared with controls . Similarly in vivo experiments were performed by microinjection of the fractionated proteins into the cytoplasm of Xenopus oocytes followed by incubation with {35S}methionine for different times . In all the experiments no evident effect of r proteins on the translation of their own mRNA was observed.

Cell, 1985 Feb, 40(2), 311 - 7
Effects of temperature and single-stranded DNA on the interaction of an RNA polymerase III transcription factor with a tRNA gene; Stillman DJ et al.; It has previously been shown that a yeast RNA polymerase III transcription factor binds stably to tRNA genes . The principal protein-DNA contacts have now been mapped, by dimethylsulfate footprinting, to the vicinity of the A and B block promoter regions of the S . cerevisiae tRNALeu3 gene . Single-stranded DNA preferentially interferes with the binding of the transcription factor to one of the promoter regions . The effect of temperature on the formation of the transcription factor-DNA complex has been examined: the complex has the kind of "opening" property that has been previously associated with prokaryotic RNA polymerase-promoter complexes, with stability increasing as the temperature is raised.

Proc Natl Acad Sci U S A, 1985 Feb, 82(4), 1266 - 70
Precise localization of genes on large animal virus genomes: use of lambda gt11 and monoclonal antibodies to map the gene for a cytomegalovirus protein family; Mocarski ES et al.; We describe an efficient procedure, which uses monoclonal antibodies directed against specific viral proteins, for the precise mapping of genes on large DNA virus genomes . We have used the technique to locate the gene encoding a family of antigenically related DNA-binding proteins on the 240-kilobase-pair human cytomegalovirus (CMV) genome . A random library of CMV DNA fragments was generated using the prokaryotic vector lambda gt11, which expresses open reading frames as beta-galactosidase fusion proteins in infected Escherichia coli . The library was screened with a mixture of monoclonal antibodies directed against the gene products of interest . The coding sequence for infected cell protein 36 (ICP36) was localized to a 2800-base-pair EcoRI fragment (map coordinates 0.228-0.240) on the CMV(Towne) and CMV(AD169) genomes by using DNA from immunoreactive lambda gt11 as probe . A 5000-nucleotide transcript from this region was detected during the early and late phases of the CMV growth cycle . This transcript directed the synthesis of the predominant member of the ICP36 family when hybrid-selected and translated in vitro . Immunoprecipitation of the in vitro translation product with the same monoclonal antibodies used in the initial mapping confirmed the location of the ICP36 gene . These studies establish the utility of the lambda gt11 expression system for rapid and precise mapping of CMV genes (or other large animal virus genes) that encode proteins for which serological reagents exist.

Proc Natl Acad Sci U S A, 1985 Feb, 82(3), 699 - 702
The first intron of the human growth hormone gene contains a binding site for glucocorticoid receptor; Moore DD et al.; Glucocorticoid receptor (GCR) protein stimulates transcription from a variety of cellular genes . We show here that GCR partially purified from rat liver binds specifically to a site within the first intron of the human growth hormone (hGH) gene, approximately 100 base pairs downstream from the start of hGH transcription . GCR binding is selectively inhibited by methylation of two short, symmetrically arranged clusters of guanine residues within this site . A cloned synthetic 24-base-pair deoxyoligonucleotide containing the predicted GCR binding sequence interacts specifically with GCR . The hGH binding site shares sequence homology with a GCR binding site upstream from the human metallothionein II gene and a subset of GCR binding sites from mouse mammary tumor virus . All of these binding sites for this eukaryotic transcriptional regulatory protein show remarkable similarity in overall geometry to the binding sites for several prokaryotic transcriptional regulatory proteins.

Arch Microbiol, 1985 Feb, 141(1), 32 - 9
Phenylalanine hydroxylase and isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase in relationship to the phylogenetic position of Pseudomonas acidovorans (Ps . sp . ATCC 11299a); Berry A et al.; The evolution of aromatic amino acid biosynthesis and its regulation is under study in a large assemblage of prokaryotes (Superfamily A) whose phylogenetic arrangement has been constructed on the criterion of oligonucleotide cataloging . One section of this Superfamily consists of a well defined (rRNA homology) cluster denoted as Group III pseudomonads . Pseudomonas acidovorans ATCC 11299a, a Group III member, was chosen for indepth studies of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase, the initial regulatory enzyme of aromatic biosynthesis . This strain is of particular interest for evolutionary studies of aromatic metabolism because it possesses phenylalanine hydroxylase, an enzyme whose physiological role and distribution among prokaryotes is largely unknown . Although P . acidovorans ATCC 11299a has been of uncertain identity, we now establish it unambiguously as a species of acidovorans by virtue of its 87% DNA homology with P . acidovorans ATCC 15668 (type strain) . This result conformed with enzyme patterning studies which placed ATCC 11299a into pseudomonad Group IIIa, a subgroup containing the acidovorans species . Crude extracts of Group III pseudomonads had previously been shown to share, as a common group characteristic, sensitivity of DAHP synthase to feedback inhibition by either L-tyrosine or L-phenylalanine . Detailed studies with partially purified preparations from strain ATCC 11299a revealed the presence of two distinct regulatory isozymes, DAHP synthase-phe and DAHP synthase-tyr . DAHP synthase-tyr is tightly controlled by L-tyrosine with 50% inhibition of activity being achieved at 4.0 microM effector . DAHP synthase-phe is inhibited 50% by 40 microM L-phenylalanine and exhibits dramatic changes in levels of activity, as well as chromatographic elution patterns, in response to dithiothreitol.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1985 Jan 25, 260(2), 1218 - 23
Processing of Bacillus cereus 569/H beta-lactamase I in Escherichia coli and Bacillus subtilis; Mezes PS et al.; The gene for Bacillus cereus 569/H beta-lactamase I, penPC, has recently been cloned and sequenced (Mezes, P . S . F., Yang, Y . Q., Hussain, M., and Lampen, J . O . (1983) FEBS Lett . 161, 195-200) . A typical prokaryotic signal peptide but with no lipoprotein modification site, as present in the Bacillus licheniformis 749/C beta-lactamase, was indicated by the DNA sequence for this secretory protein . We have here purified the beta-lactamase I products found in Escherichia coli and Bacillus subtilis carrying penPC and have determined the first 20 NH2-terminal amino acids of each of the forms . Processing of the beta-lactamase I in E . coli occurs at a single site which is characteristic for cleavage by a signal peptidase . B . subtilis secreted two distinct products to the culture medium which were both smaller than the single product formed in E . coli . Sequencing of {35S}Met-labeled pre-beta-lactamase I from phenylethyl alcohol-treated cells of B . cereus 569/H indicated that UUG is being utilized as the initiation codon for penPC . The same result was obtained for the pre-beta-lactamase I from similarly treated cells of the closely related B . cereus 5/B strain.

Nature, 1985 Jan 24-30, 313(6000), 323 - 5
G to A substitution in the distal CCAAT box of the A gamma-globin gene in Greek hereditary persistence of fetal haemoglobin; Gelinas R et al.; The sequence 5'TTGGPyCAAT 3' (the 'CCAAT box') is a constituent of the promoter region of many eukaryotic and prokaryotic genes and is believed to play a part in promoter function . A characteristic of the two fetal human globin genes (A gamma and G gamma) is a duplication of a 12-base pair (bp) sequence containing the CCAAT box . Here we report a G----A substitution in the TTG sequence of the distal CCAAT box of the A gamma-globin gene in an individual with the A gamma (Greek) type of hereditary persistence of fetal haemoglobin (HPFH) . This represents the first report of a natural mutation of the CCAAT box in a eukaryotic gene . The fact that this transition is associated with inappropriate expression of the A gamma gene in adult life suggests that the CCAAT box (or its surrounding sequences) may have a role in the developmental control of gamma-globin genes.

J Theor Biol, 1985 Jan 21, 112(2), 433 - 44
A three-dimensional representation for base composition of protein-coding DNA sequences; Rowe GW; Multi-dimensional scaling is applied to our codon space data on the protein coding sequences of DNA from a wide variety of organisms in an attempt to find the smallest number of parameters which will accurately represent these sequences . I find that a three-dimensional representation is satisfactory . One of the three resulting co-ordinates separates eukaryotes and their associated viruses from prokaryotes and their associated phages, while an orthogonal co-ordinate separates those organisms capable of synthesizing proteins (eukaryotes and prokaryotes) from those not so capable (viruses and phages) . Mitochondria show no relation in our plots to any of these groups.

Nature, 1985 Jan 17-23, 313(5999), 243 - 6
A retrovirus-like strategy for expression of a fusion protein encoded by yeast transposon Ty1; Mellor J et al.; Eukaryotic transposons such as the Ty element of yeast or the copia-like sequences of Drosophila show structural and functional similarities to both prokaryotic transposons and retroviral proviruses, but the prokaryotic transposons and retroviral proviruses use markedly different expression strategies which yield products having entirely different functions . To determine the phylogenetic relationship between eukaryotic transposons, prokaryotic transposons and retroviruses, we have sought to identify and characterize the proteins encoded by the yeast Ty element and to describe the strategies used to express these proteins . We show here that the yeast transposon produces a fusion protein by a specific frameshifting event that fuses two out-of-phase open reading frames (ORFs) . The process is remarkably similar to that used by retroviruses such as Rous sarcoma virus (RSV) to produce Pr180gag-pol.

Nucleic Acids Res, 1985 Jan 11, 13(1), 185 - 94
A method to locate protein coding sequences in DNA of prokaryotic systems; Kolaskar AS et al.; cDNA sequence data from E . coli phages, for which complete genome sequences are known, have been analysed, From this analysis thirteen triplets have been identified as markers to distinguish protein-coding frames from fortuitous open reading frames . The region of -18 to +18 nucleotides around ATG/GTG, has been analysed and used to identify initiator codons from internal ATG/GTG . With the aid of criteria defined above a method has been developed to locate protein coding sequences by a combination of 'gene search by signal' and 'gene search by content' approaches . Application of this method to prokaryotic systems including those which were not part of our data base indicates that it is quite accurate and general in nature.

Mutat Res, 1985 Jan-Feb, 148(1-2), 107 - 12
Induction by modified purines (2-aminopurine and 6-N-hydroxylaminopurine) of chromosome aberrations and aneuploidy in Syrian hamster embryo cells; Tsutsui T et al.; The modified purines, 2-aminopurine and 6-N-hydroxylaminopurine, are known point mutagens in prokaryotic organisms . 2-Aminopurine is much less potent than 6-N-hydroxylaminopurine in inducing gene mutation in mammalian cells in culture and this corresponds to the relative activity of these two compounds in inducing tumors in rats and neoplastic transformation of Syrian hamster embryo cells in culture . We report here that these modified purines can induce chromosome aberrations, including chromatid gaps, breaks, and exchanges, as well as numerical chromosome changes in Syrian hamster embryo cells . These chromosome mutations occur over the concentration range of chemical needed to induced morphological transformation of the same cells . It is not known how nucleic base analogs induce chromosome mutations; however, this activity must be considered in attempting to understand the mechanism by which these agents induce neoplastic transformation of cells.

J Mol Evol, 1985, 22(4), 363 - 5
Codon usage and genome composition; Bernardi G et al.; The GC levels of codon third positions from 49 genomes covering a wide phylogenetic range are linearly correlated with the GC levels of the corresponding genomes . Three different relationships have been found: one for prokaryotes and viruses, one for lower eukaryotes, and one for vertebrates . All points not fitting the first relationship can be brought into quasi coincidence with it when plotted against GC levels of coding sequences.

Biosystems, 1985, 18(3-4), 279 - 92
Glucanase gene diversity in prokaryotic and eukaryotic organisms; Mackay RM et al.; A number of bacteria and eukaryotes produce extracellular enzymes that degrade various types of polysaccharides including the glucans starch, cellulose and hemicellulose (xylan) . The similarities in the modes of expression and specificity of enzyme classes, such as amylase, cellulose and xylanase, suggest common genetic origins for particular activities . Our determination of the extent of similarity between these glucanases suggests that such data may be of very limited use in describing the early evolution of these proteins . The great diversity of these proteins does allow identification of their most highly conserved (and presumably functionally important) regions.

J Mol Evol, 1985, 22(1), 32 - 8
Phylogenetic relationships among eukaryotic kingdoms inferred from ribosomal RNA sequences; Hasegawa M et al.; Phylogenetic trees among eukaryotic kingdoms were inferred for large- and small-subunit rRNAs by using a maximum-likelihood method developed by Felsenstein . Although Felsenstein's method assumes equal evolutionary rates for transitions and transversions, this is apparently not the case for these data . Therefore, only transversion-type substitutions were taken into account . The molecules used were large-subunit rRNAs from Xenopus laevis (Animalia), rice (Plantae), Saccharomyces cerevisiae (Fungi), Dictyostelium discoideum (Protista), and Physarum polycephalum (Protista); and small-subunit rRNAs from maize (Plantae), S . cerevisiae, X . laevis, rat (Animalia), and D . discoideum . Only conservative regions of the nucleotide sequences were considered for this study . In the maximum-likelihood trees for both large- and small-subunit rRNAs, Animalia and Fungi were the most closely related eukaryotic kingdoms, and Plantae is the next most closely related kingdom, although other branching orders among Plantae, Animalia, and Fungi were not excluded by this work . These three eukaryotic kingdoms apparently shared a common ancestor after the divergence of the two species of Protista, D . discoideum and P . polycephalum . These two species of Protista do not form a clade, and P . polycephalum diverged first and D . discoideum second from the line leading to the common ancestor of Plantae, Animalia, and Fungi . The sequence data indicate that a drastic change occurred in the nucleotide sequences of rRNAs during the evolutionary separation between prokaryote and eukaryote.

Gene, 1985, 37(1-3), 145 - 54
Effects of heterologous ribosomal binding sites on the transcription and translation of the lacZ gene of Escherichia coli; Looman AC et al.; A vector (pKL203) was constructed which contains the promoter-operator region of the lacZ gene and the major part of the coding sequence of the lac operon . The lacZ translation initiation signals {Shine-Dalgarno (SD) sequence and AUG codon} were deleted, and in their place a synthetic linker sequence was inserted, providing single restriction sites for SmaI and BamHI . With this vector constructions were made in which initiation signals of other prokaryotic genes (phage MS2 maturation protein, phage Q beta A2 gene and tufB gene) were fused to the lacZ gene, giving rise to various fusion proteins . The introduction of N-terminal amino acids (aa) in beta-galactosidase (beta-gal) which differ from the wild-type aa invariably leads to an enzyme with a strongly reduced thermostability as compared to the wild-type enzyme . Therefore an immunoprecipitation method was used to measure the amount of fusion protein . It was found that these amounts varied strongly from one construction to another . Concomitant determinations of the amounts of lac-operon-specific mRNA showed an unexpectedly large variation among the clones . No strict correlation could be found between the level of lac mRNA and beta-gal production . Per molecule of lac mRNA, translation appears to be most efficient when the homologous lacZ initiation signal is present.

C R Acad Sci III, 1985, 300(9), 377 - 80
{Localization of initiating codons in RNA prokaryotes messengers by learning technics}; Rodier F et al.; Learning processes are applied to the recognition of protein coding regions in prokaryotes . Non-contradictory, statistical rules are deduced from a set of known examples of coding regions . These rules allow us to build characteristic patterns on the m-RNA upstream the initiating codon . These rules are applied to recognize more than 180 coding sequences.

Cancer Surv, 1985, 4(3), 517 - 28
Direct and inducible mutagenesis in mammalian cells; Summers WC et al.; The understanding of mutagenic mechanisms in mammalian cells is based on extrapolations of results obtained in prokaryotes and simple eukaryotes . The use of animal viruses as targets for mutagenesis suggests that these extrapolations may be valid: recent data indicate that mutagenesis of ultraviolet-damaged templates in mammalian cells seems to occur at similar sites to that observed in prokaryotes; furthermore, nuclear replicating animal viruses are subject to the phenomenon of ultraviolet-enhanced reactivation, called SOS reactivation in bacteria . In experiments analogous to Weigle mutagenesis of phage, several groups have shown that animal cells appear to respond to DNA damage by induction of mutagenic pathways which can act upon infecting viral genomes . Recent experiments with shuttle vectors that can replicate both in animal cells and in bacteria have confirmed these conclusions . These vectors now make possible the rapid recovery of mutant genes for direct sequence analysis.

Adv Biophys, 1985, 19, 91 - 131
Protein structures and split genes; Go M; Exon-intron structures of eukaryotic genes were examined closely in their relation to primary and tertiary structures of the proteins they encode . Specific attention was given to the introns of genes encoding proteins having no repeats in their amino acid sequences . such introns have been shown to be located at sites corresponding to inter-domain or inter-module junctions of proteins identified in their three dimensional structures . "Modules," compact structural units in globular domains of proteins, are identified by drawing a distance map . Intron positions are found to correspond to intermodule junctions in various proteins whose X-ray crystallographic data are available: the globin family, CEWL, ovomucoid, cytochrome c, ADH, and trypsin-like serine proteinases . The good correspondence between intron positions and intermodule junctions excludes a mechanism of random insertion of introns, because the probability of intron insertion at each intermodule junction is extraordinarily small . Intron positions have been very stable and well conserved during evolution . However, at some inter-module junctions no introns are found . Modules in small proteins having no core modules buried in their interior have a character suitable for recruitment through their assembly into a stable domain; one side of them is rich in hydrophobic residues and the other in hydrophilic residues . Functionally important residues are scattered on different modules in the proteins examined . Based on these observations, the role of modules in the precellular period was conjectured: some of them might be functionally active by themselves but most modules might be only segments who could function as an active protein only in an assembly . The origin of introns might be traced back prior to the divergence of prokaryotes and eukaryotes.

Biosystems, 1985, 18(3-4), 307 - 19
Molecular organization of dinoflagellate ribosomal DNA: evolutionary implications of the deduced 5.8 S rRNA secondary structure; Maroteaux L et al.; The 5.8 S rRNA gene of Prorocentrum micans, a primitive dinoflagellate, has been cloned and its 159 base pairs (bp) have been sequenced along with the two flanking internal transcribed spacers (ITS 1 and 2), respectively, 212 and 195 bp long . Nucleotide sequence homologies between several previously published 5.8 S rRNA gene sequences including those from another dinoflagellate, an ascomycetous yeast, protozoans, a higher plant and a mammal have been determined by sequence alignment . Two prokaryotic 5'-ends of the 23 S rRNA gene have been compared owing to their probable common origin with eucaryotic 5.8 S rRNA genes . Several nucleotides are distinctive for dinoflagellates when compared with either typical eucaryotes or procaryotes . This is consistent with an early divergence of the dinoflagellate lineage from the typical eucaryotes . The secondary structure of dinoflagellate 5.8 S rRNA molecules fits the model of Walker et al . (1983) . Conserved nucleotides which distinguish dinoflagellate 5.8 S rRNA from that of other eucaryotes are located in specific loops which are assumed to play a structural role in the ribosome . A 5.8 S rRNA phylogenetic tree which is proposed, based on sequence data, supports our initial assumption of the dinoflagellates.

Biosystems, 1985, 18(3-4), 249 - 62
Histones in protistan evolution; Rizzo PJ; The potential of comparative studies on histones for use in protistan evolution is discussed, using algal histones as specific examples . A basic premise for the importance of histones in protistan evolution is the observation that these proteins are completely absent in prokaryotes (and cytoplasmic organelles), but with few exceptions, the same five major histone types are found in all higher plants and animals . Since the histone content of the algae and other protists is not constant, some of these organisms may represent transition forms between the prokaryotic and eukaryotic modes of packaging the genetic material . Comparative studies of protistan histones may thus be of help in determining evolutionary relationships . However, several problems are encounter with protistan histones, including difficulties in isolating nuclei, proteolytic degradation, anomalous gel migration of histones, and difficulties in histone identification . Because of the above problems, and the observed variability in protistan histones, it is suggested that several criteria be employed for histone identification in protists.

Biosystems, 1985, 18(3-4), 223 - 40
The prokaryote-eukaryote interface; Hunt LT et al.; Over the past 30 years the study of the sequences of proteins and nucleic acids has produced almost incredible amounts of information, new concepts, and new avenues of research . The beginning was slow: the first peptide hormones sequenced in the early 1950's, the first cytochrome c (horse) in 1961, the first bacterial ferredoxin in 1964, and the first transfer RNA (yeast alanine tRNA) in 1965 . In the past 6 years, the rate of data accumulation has accelerated tremendously, primarily due to technological advances in nucleic acid sequencing techniques . For investigators of biological evolution, the sequence data and the new information on genetic mechanisms would prove to be the best evidence for elucidating relationships among the genomes of living organisms and for deducing phylogenetic history . In particular, they needed evidence to decide between the two hypotheses for the origin of eukaryotic cells . Now, less than 20 years since Margulis renewed the investigation of this problem, comparisons of protein and nucleic acid sequences, especially of the small subunit ribosomal RNAs, have answered this question in favor of the endosymbiotic origin of eukaryotic cells . After briefly discussing some of the concepts that helped resolve this controversy and the problems involved in using sequence data for evolutionary studies, we describe a few examples of useful evolutionary trees.

Radiat Environ Biophys, 1985, 24(4), 281 - 6
Photoreactivation of damage induced by ionizing radiation in yeast cells; Petin VG et al.; Experimental data on photoreactivation of damage induced by ionizing radiation in yeast cells are presented . The value of photoreactivation was found to be the highest for the following conditions predicted by us as optimum ones: large volume of irradiated suspension, hypoxia and high energy sparsely ionizing radiation . A comparison of data for yeast and bacterial cells shows that Cerenkov emission from ionizing radiation may produce photoreactivated pyrimidine dimers in both prokaryotic and eukaryotic cell systems.

Annu Rev Microbiol, 1985, 39, 21 - 50
Mechanisms of bacterial virulence; Brubaker RR; In this review the nature of prokaryotic parasites was first discussed with emphasis on the evolution of virulence . Subsequently, nonspecific mechanisms of host defense were considered with emphasis on recent findings relating to bacterial killing by serum and professional phagocytes . Based on this background, the nature of virulence factors required for growth of pathogens in the nonimmune host was considered . Strategies used by extracellular and intracellular parasites were compared . It is evident from the resulting overview of experimental findings that knowledge concerning virulence of extracellular parasites outweighs that collected for both facultative and obligate intracellular parasites . Remaining problems regarding extracellular parasitism include precise resolution of the nature of serum resistance, pilus-independent adhesion, tissue invasiveness, and resistance to phagocytosis . Solutions to these questions will probably arise during the course of studies primarily emphasizing bacterial structure and function . Unresolved problems concerning intracellular parasites include definition of regulatory changes involved in adaptation for intra- and extracellular growth, the nature of reactions preventing phagosome-lysosome fusion, mechanisms of survival within phagolysosomes, and explanations for host-cell dependence . These topics provide real problems in cellular and molecular biology, and they will probably be resolved by those familiar with these disciplines . The ability of parasitic prokaryotes to shut off otherwise effective specific immune responses was shown to cross phenotypic lines . Resolution of these somewhat sinister mechanisms of virulence will require an understanding of fundamental immune processes . Further study of bacterial virulence factors will probably provide an understanding of basic cellular processes relevant to other biological disciplines . Indeed, information of this nature may not be obtainable by any other experimental approach.

Gene, 1985, 35(1-2), 1 - 9
Expression of a nitrogen-fixation gene encoding a nitrogenase subunit in yeast; Berman J et al.; Biological nitrogen fixation is catalyzed by nitrogenase, an enzyme complex exclusive to prokaryotes . We used the yeast Saccharomyces cerevisiae to study the synthesis, and subsequently the assembly, of nitrogenase components in a eukaryote . Here, the Klebsiella pneumoniae nifH gene, encoding the subunit of the Fe protein (Kp2) component of nitrogenase, was expressed in S . cerevisiae from the yeast ADHI promoter . The nifH gene product, detected in yeast by immunoblot analysis with anti-Kp2 antibodies, exhibited the same electrophoretic mobility in SDS-polyacrylamide gels as that of the Kp2 subunit synthesized in K . pneumoniae . Estimates of Kp2 antigen and assays of beta-galactosidase activity specified by nifH'-'lacZ fusions showed that the level of nifH product was similar in anaerobically and aerobically grown yeast, but varied with different transforming plasmids and in various haploid and diploid yeast strains . A cistron located downstream to nifH in a transcript resembling the polycistronic mRNA of the nifHDKY operon in K . pneumoniae is not translated in yeast.

Z Parasitenkd, 1985, 71(4), 435 - 42
Electron microscope observations on the interaction of Mycoplasma fermentans with Trichomonas vaginalis; Scholtyseck E et al.; Cultures of Trichomonas vaginalis were found to be contaminated with Mycoplasma fermentans . By means of electron microscopy the interaction between the prokaryotic organisms and the trichomonads was examined . Cells of M . fermentans were observed in the medium; some of them were attached to the surface of the trichomonads and others were observed in membrane-bounded vacuoles of trichomonads . They were also present in the ground substance of the cytoplasm . The mycoplasmas divided by binary fission like other prokaryotes . The most obvious change occurring in the infected trichomonad cells was an increase in number of vacuoles containing mycoplasmas.

Biosystems, 1985, 17(3), 209 - 25
tRNA-rRNA sequence homologies: evidence for an ancient modular format shared by tRNAs and rRNAs; Bloch DP et al.; Homologies between tRNAs and rRNAs are identified in searches using various combinations of Escherichia coli, yeast, Halobacterium volcanii and bovine mitochondrial sequences . As in previously reported comparisons, the homologies are too frequent and long to be attributed to coincidence, and similar frequencies from inter- and intraspecies comparisons preclude evolutionary convergence as an explanation . In contrast to the earlier studies, patterns in the positioning of the homologies are now described . Graphing the positions of the homologies along orthogonal axes that represent numbers of bases in tRNA and rRNA shows recurring patterns in the alignments . Preferred spacings of integral multiples of 9 bases are found, suggesting a periodicity in the ancestral structure from which the tRNAs and rRNAs were derived . The periodicity also suggests persistence of a modular format in both classes of molecules that survived changes in sequence that occurred during evolution . A model is proposed for the generation of the ancestral molecule and the early evolution of the coding mechanism . Elongation by self-priming and self-templating gave a hairpin with a 9 base stem . Two additional cycles gave a 70-80 base tRNA-like structure . Additional cycles yielded a tandem repeat of this unit, roughly equivalent in size to the combined rRNAs of prokaryotes . The larger RNA would contain the information and materials for generating the smaller RNAs . It is proposed that multiple recombination among such molecules gave composite structures, presumed progenitors of today's t- and rRNAs . The distribution of the conserved domains among today's species argues for the existence of the ancestral molecule prior to divergence of lines leading to the various kingdoms . Their presence in the different nucleic acids suggests the existence of a nucleic acid with multiple functions prior to partitioning of these functions among the nucleic acids that exist today . The occurrence of overlaps, overlays and consensus alignments among the homologies provides the means for identifying contiguous and neighboring conserved regions and holds promise for the reconstruction of the sequence of an ancestral molecule.

Mol Biochem Parasitol, 1985 Jan, 14(1), 29 - 40
Reduction of nitroimidazole derivatives by hydrogenosomal extracts of Trichomonas vaginalis; Yarlett N et al.; The reduction rate of nitroimidazole derivatives by pyruvate:ferredoxin oxidoreductase activity in ferredoxin depleted hydrogenosomal extracts of Trichomonas vaginalis depended on the one-electron midpoint potential (E7(1)) of 15 compounds out of the 16 tested . The results showed a linear correlation with a positive slope between the logarithm of the rate and the E7(1) in the range from -564 to -260 mV . Addition of T . vaginalis ferredoxin stimulated the reduction . The additional rate (stimulated rate minus basal rate) was proportional to the concentration of ferredoxin and independent of the E7(1) of the compounds . The compound with the most positive E7(1) (-243 mV) was, however, reduced more slowly than expected . These findings indicate that reduction in the presence of ferredoxin is the sum of two processes, i.e . electron transfer directly from pyruvate:ferredoxin oxidoreductase and from reduced ferredoxin generated by pyruvate:ferredoxin oxidoreductase activity . The relative role of ferredoxin in reductive activation of nitroimidazole derivatives is greater for compounds with more negative E7(1) values . This observation correlates with the high selectivity of the more negative 5-nitroimidazoles against anaerobic prokaryotic and eukaryotic microorganisms in which ferredoxin plays an important metabolic role.

Curr Top Cell Regul, 1985, 26, 455 - 68
Regulation of antibiotic resistance in bacteria: the chloramphenicol acetyltransferase system; Shaw WV et al.; The evaluations of antibiotic resistance has been a subject of interest to workers in several disciplines . Our current understanding of the molecular biology of plasmids, phages, and transposable elements provides a basis for appreciating the range of mechanisms likely to be involved in the horizontal spread of resistance determinants through microbial ecosystems . Rather less can be imagined with confidence about the origins of the genes or the constraints and selection pressures operating at the level of protein structure . The CAT system illustrates the extent of variation possible for an accessory gene product which is required infrequently and which is encoded by multicopy and promiscuous vectors which can cross taxonomic boundaries . Still less is known with certainty about the evolution of genetic control of the expression of antibiotic resistance . While there are sound reasons for looking in detail at prokaryotic antibiotic-producing organisms such as Streptomyces to find the progenitors of present resistance mechanisms (44, 45), it seems likely that controls of expression have been acquired during the "passage" of selectable markers through more distant bacterial genera . The CAT system is illustrative of the variety we may expect to find in control strategies used by microbial systems generally . It might indeed be a surprise to find an expression mechanism operating in the CAT system (or for any other family of resistance genes) which was not illustrative of a general strategy exploited by essential genes specifying biosynthetic or degradative functions . There may be some truth in referring to the cat structural gene as a "cartridge" for the isolation and manipulation of promoter functions . It would seem that nature has been at it for some time.

Proc Natl Acad Sci U S A, 1985 Jan, 82(1), 19 - 23
Eukaryotic transient expression system dependent on transcription factors and regulatory DNA sequences of vaccinia virus; Cochran MA et al.; A transient expression system in which chimeric genes are expressed in cells infected with vaccinia virus was developed . Recombinant plasmids containing the promoter regions of vaccinia virus genes ligated to the coding segment of the prokaryotic chloramphenicol acetyltransferase (CAT) gene were constructed . When the plasmids were introduced into vaccinia virus-infected cells by transfection, the chimeric gene was expressed and significant levels of CAT accumulated . CAT activity was not detected when the same recombinant plasmid was introduced into uninfected cells, nor was activity detected when the vaccinia virus promoter was absent from the plasmid or was replaced by simian virus 40 or Rous sarcoma virus promoters . This specificity indicated that expression is dependent on a cis-acting vaccinia virus promoter region within the recombinant plasmid and diffusible trans-acting transcription factors produced during virus infection . The lack of effect of a simian virus 40 enhancer element inserted upstream of the vaccinia virus promoter region also distinguished this system from systems dependent on RNA polymerase II . Although replication of the recombinant plasmid could not be detected in either uninfected or vaccinia virus-infected cells, an inhibitor of DNA synthesis significantly reduced CAT expression . This result, as well as the kinetics of CAT synthesis, suggests that replication of viral DNA templates can enhance transcription of chimeric genes in recombinant plasmids.

Adv Enzyme Regul, 1985, 23, 193 - 215
The biochemical pharmacology of CI-920, a structurally novel antibiotic with antileukemic activity; Jackson RC et al.; CI-920 is a structurally novel, phosphate-containing polyene lactone antitumor agent isolated from a previously undescribed subspecies of Streptomyces pulveraceus cultured from a Brazilian soil sample . CI-920 was active against murine leukemia P388, and highly active and curative against L1210 leukemia in vivo . CI-920 was less active or inactive against the murine solid tumors tested . Daily administration for five to nine days was more effective against L1210 leukemia than a single dose or doses every four days . Given three times daily for five days, CI-920 was more toxic and less active . CI-920 had similar activity intravenously and intraperitoneally . Oral administration was inactive and nontoxic . Subcutaneous treatment was less effective and more toxic . Structure-activity relationship studies showed that the phosphate group was essential for antitumor activity in vivo and in vitro . Hydrolyzing the lactone ring also resulted in loss of antitumor activity, as did acetylation of the 6-hydroxyl group . Hydroxylation at the 5-position of the lactone ring resulted in partial retention of antitumor activity, but in greater toxicity to mice . Removal of the 13-hydroxyl group resulted in retention of high antitumor activity with approximately three-fold improvement in dose-potency . CI-920 is not cytotoxic to prokaryotic cells . CI-920 causes inhibition of biosynthesis of RNA and DNA in intact L1210 cells . Protein synthesis is also inhibited at higher drug concentrations . The inhibition of nucleic acid synthesis is not an antimetabolite effect, since pools of ribonucleoside triphosphates and deoxyribonucleoside triphosphates are not depleted . CI-920 does not cause DNA strand breakage, as measured by alkaline elution, and is not mutagenic in the Ames test at concentrations up to 200 micrograms/ml . CI-920 does not cause direct inhibition of RNA polymerase or DNA polymerase in permeabilized cells . It is possible that CI-920 must be metabolically activated within the target cells; alternatively it may interact with a component of chromatin other than DNA or the polymerases . Flow cytometry studies showed that growth-inhibitory levels of CI-920 caused accumulation of cells in the G2+M region . Higher drug concentrations caused an S-phase block . CI-920 is an inhibitor and irreversible inactivator of reduced folate membrane transport, and appears to enter cells by this receptor . L1210 cells selected for resistance to CI-920 are cross-resistant to methotrexate, and deficient in reduced folate transport.(ABSTRACT TRUNCATED AT 400 WORDS)

Gene, 1985, 37(1-3), 171 - 9
Translational interference at overlapping reading frames in prokaryotic messenger RNA; Berkhout B et al.; In overlapping reading frames of prokaryotic mRNA, the ribosome-binding site (RBS) of the downstream cistron is part of the coding sequence of the upstream message . We have examined whether the rate of translation in Escherichia coli can be sufficiently high to preclude the use of an RBS in initiation of protein synthesis when it is part of an actively decoded reading frame . The two sets of gene overlap present in the RNA phage MS2 are used as a model system . We find that translation of an upstream cistron can fully block initiation of protein synthesis at the overlapping RBS of the downstream cistron . Nonsense mutations in the upstream gene restore the translation of the downstream gene.

Acta Biochim Biophys Acad Sci Hung, 1985, 20(3-4), 203 - 11
Liposome mediated DNA-transfer into mammalian cells; Somlyai G et al.; We have investigated the interaction of mammalian cells with liposome encapsulated DNA . Tissue cultured mammalian cells were exposed to large, unilamellar phosphatidyl serine liposomes containing DNA molecules from different animal cells or prokaryotic organisms . The liposomes bind rapidly to the surface and are taken up by the cells and significant proportion of the encapsulated DNA is transported to the nuclei . Transient expression of the foreign genetic material could be detected in high percentage of the treated cells for a few days . During this period of time foreign DNA is present in both free and integrated form, however, the free form soon disappears . Stable transformant cell colonies--with continuous expression of new gene(s)--were isolated under selective pressure with a frequency of approx . 10(-5).

Gene, 1985, 40(2-3), 183 - 90
"ATG vectors' for regulated high-level expression of cloned genes in Escherichia coli; Amann E et al.; A plasmid cloning vector system has been constructed that allows for the production of large quantities of foreign proteins or fragments thereof, in an unfused state . These vectors provide strong regulated trp-lac fusion promoters and the lacZ ribosome-binding site (RBS) followed by an ATG translation initiation codon at an appropriate distance from the RBS . The ATG codon is located within a unique NcoI restriction site (CCATGG) . Digestion with NcoI exposes the ATG for fusion . Gene fragments lacking a prokaryotic RBS and/or ATG start codons can be inserted in several ways . Expression experiments using a truncated cI gene of bacteriophage lambda or a large portion of the coding region of the Herpes simplex virus type 1 glycoprotein D gene have been performed . The results of these studies show that the vectors are useful for the high-level expression of prokaryotic and eukaryotic genes in Escherichia coli.

Mol Gen Genet, 1985, 201(2), 198 - 203
Gene organization and target specificity of the prokaryotic mobile genetic element IS26; Mollet B et al.; The 820-bp mobile genetic element IS26 loses its ability to promote transpositional cointegration (1) by short deletions near the middle of the element causing shifts in both reading frames ORFI (left to right) and ORFII (right to left) and (2) by deletions causing substitutions of the C-terminus of ORFI but not affecting ORFII . The 702-bp ORFI is thus likely to code for the IS26 transposase . An 82-bp long sequence from the left end of IS26 contains a promoter-like structure in front of the start of ORFI at coordinate 64 . In appropriately constructed plasmids, this sequence promotes the expression of the galK structural gene . The observation provides additional evidence for the functional relevance of ORFI . Neither the presence nor the absence of an intact IS26 element on the same plasmid affects measurably the degree of the galK gene expression by the IS26 promoter . Sequence comparison of 14 independent integration sites of IS26 and its relatives reveals no striking rules for target selection by the element, and the distrubtion of integration sites of IS26 on small multicopy plasmids is nearly random and independent of the local AT-content.

Gene, 1985, 38(1-3), 31 - 8
Versatile expression vectors for high-level synthesis of cloned gene products in Escherichia coli; Crowl R et al.; We have constructed a set of expression vectors which contain synthetic DNA sequences comprising a computer-generated model ribosomal binding site located downstream from the tightly regulated phage lambda pL . promoter . These vectors have been used in several laboratories to produce significant amounts of eukaryotic and prokaryotic gene products in Escherichia coli, either as fusion proteins (with two to nine extra N-terminal amino acids) or as proteins containing the naturally occurring amino terminus . For inserting DNA sequences downstream of an initiation codon, we used synthetic oligonucleotides to introduce multiple-use restriction sites recognized by EcoRI, BamHI and ClaI which generate termini complementary to those of a variety of enzymes (e.g., EcoRI, MboI, TaqI, and HpaII), in addition to their own . A set of three of these vectors was made to accommodate all three translational reading frames . In combination, the features of these vectors afford useful advantages over expression vectors previously described, especially for the application of shot-gun cloning of genomic DNA to generate expression libraries.

Gene, 1985, 38(1-3), 215 - 26
Cloning and analysis of the promoter region of the erythromycin resistance gene (ermE) of Streptomyces erythraeus; Bibb MJ et al.; A DNA fragment containing the coding and regulatory sequences of the erythromycin (Er) resistance (ermE) gene of the Er produces Streptomyces erythraeus was cloned in Streptomyces lividans using the plasmid vector pIJ61 . The approximate location and orientation of ermE were deduced from studies of its expression after subcloning in Escherichia coli . Sequences responsible for transcription of ermE in Streptomyces were studied by nucleotide (nt) sequencing, high resolution S1 and exonuclease VII mapping, in vitro transcription and in vivo promoter-probing . Tandemly arranged promoters of typical prokaryotic appearance initiate transcription of the coding region of ermE; a promoter of similar sequence was identified that initiates transcription of a likely coding region running in the opposite direction to ermE . It is suggested that these sites represent a class of vegetatively expressed Streptomyces promoter that is utilised by a form of RNA polymerase holoenzyme that also recognizes typical promoters of other bacterial genera.

Basic Life Sci, 1985, 34, 157 - 82
Polysubstrate monooxygenases in Drosophila, mammals and man; Wurgler FE; There is overwhelming evidence that polysubstrate monooxygenases play a central role in the metabolism of endogenous compounds as well as in the biotransformation of xenobiotics . These enzyme systems are of great importance in such diverse fields as insecticide resistance, mutagenicity, carcinogenicity, drug metabolism, etc . The constitutive and, in particular, the induced forms represent various products from a multigene family . This has first been shown for the mouse, but evidence is accumulating that this is also true for other mammals and for man . Also in insects a similar picture is emerging . If the regulation of cytochrome P-450 induction resembles in any way the other methods by which prokaryotes and eukaryotes cope genetically with the many forms of environmental selective pressures, it is very likely that most organisms have the genetic capacity to produce not only hundreds but probably thousands of inducible forms of cytochrome P-450 (Nebert et al., 1981) . Doubtless, many fields from pest control to cancer prevention to drug safety will profit from the elucidation of the genetic mechanisms involved.

CRC Crit Rev Biochem, 1985, 18(4), 327 - 83
Attenuation may regulate gene expression in animal viruses and cells; Aloni Y et al.; In eukaryotes, an abundant population of promoter-proximal RNA chains have been observed and studied, mainly in whole nuclear RNA, in denovirus type 2, and in SV40 . On the basis of these results it has been suggested that a premature termination process resembling attenuation in prokaryotes occurs in eukaryotes . Moreover, these studies have shown that the adenosine analog 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) enhances premature termination, but its mode of action is not understood . The determination of the nucleotide sequences of SV40 and other viruses and cellular genes provide means for elucidating the nucleotide sequences involved in the attenuation mechanism . A model has recently been described in which attenuation and mRNA modulation in a feedback control system quantitatively regulate SV40 gene expression . The suggested mechanism described in this model opens up approaches to the investigation of attenuation and mRNA modulation as a possible mechanism whereby eukaryotes may regulate transcription in a variety of different circumstances.

Mol Gen Genet, 1985, 199(3), 534 - 6
An active variant of the prokaryotic transposable element IS903 carries an amber stop codon in the middle of an open reading frame; Mollet B et al.; The prokaryotic mobile genetic element IS903.B is an active variant of IS903 . It differs from IS903 and IS102 by 34 and 61 nucleotide substitutions, respectively . The large open reading frame (ORFI) which probably encodes the transposase is conserved in all three IS elements, whereas the smaller open reading frame (ORFII), which codes on the opposite DNA strand and entirely overlaps ORFI, contains an amber stop codon past the middle of ORFII in IS903.B . Experiments using Escherichia coli K12 strains permissive or non-permissive for amber mutations revealed no difference in the cointegration frequency mediated by IS903.B . Therefore, a possible peptide encoded by ORFII on the IS903-related element is unlikely to be necessary for transposition.

Basic Life Sci, 1985, 30, 469 - 87
Mechanisms essential for stable inheritance of mini-F plasmid; Hiraga S et al.; The F plasmid has its own partition mechanism and ccd mechanism (coupled cell division), besides its own replication mechanism, in order to be stably inherited into daughter cells through cell division . These 3 mechanisms are independent of one another . Therefore, when a DNA segment essential and sufficient for a mechanism is jointed to other heterologous plasmids, the segment is also functional . Most of natural low copy number plasmids might also have their own replication, partition, and ccd mechanisms . These 3 mechanisms may be fundamental to ensure stable inheritance for low copy-number replicons in prokaryotes.

J Mol Appl Genet, 1985, 3(1), 36 - 44
Structure of the penicillin acylase gene from Escherichia coli: a periplasmic enzyme that undergoes multiple proteolytic processing; Bruns W et al.; Penicillin acylase is processed from a 90-kD precursor through the cleavage of a leader peptide and two further endopeptidase cleavages to yield an enzyme that contains a 22-kD (or 23-kD) and a 65-kD subunit . The endopeptidase cleavages require an intact carboxy terminus . This type of processing appears to be unique for a prokaryotic enzyme, having its most closely related analog in the synthesis and processing of preproinsulin and other eukaryotic hormones.

Biomed Biochim Acta, 1985, 44(2), 235 - 41
Characterization of the DNA of the hamster papovavirus: II . A comparison of binding sites for Escherichia coli- and calf thymus RNA polymerase II on the hamster papovavirus genome; Vogel F et al.; Calf thymus (CT) RNA polymerase II bound to hamster papovavirus (HaPV) DNA was visualized by electron microscopy and compared to binding sites obtained after binding of Escherichia coli RNA polymerase to the HaPV genome . Thirteen binding sites were observed with CT polymerase II at map positions 0.04-0.07; 0.11; 0.18; 0.30; 0.37; 0.47; 0.57; 0.65; 0.78; 0.83; 0.90 and 0.94 using the unique BamHI cleavage site as zero point on the HaPV physical map . These binding sites correlate well with A + T rich sequences within the HaPV DNA as revealed by experiments using protein 32 coded for by phage T4 . A comparison of binding of prokaryotic and eukaryotic RNA polymerase demonstrates a high degree of correspondence for most of the binding sites on the HaPV genome.

Genetics, 1985 Jan, 109(1), 3 - 19
Mismatch repair mutations of Escherichia coli K12 enhance transposon excision; Lundblad V et al.; Excision of the prokaryotic transposon Tn10 is a host-mediated process that occurs in the absence of recA function or any transposon-encoded functions . To determine which host functions might play a role in transposon excision, we have isolated 40 mutants of E . coli K12, designated tex, which increase the frequency of Tn10 precise excision . Three of these mutations (texA) have been shown to qualitatively alter RecBC function . We show that 21 additional tex mutations with a mutator phenotype map to five genes previously identified as components of a methylation-directed pathway for repair of base pair mismatches: uvrD, mutH, mutL, mutS and dam . Previously identified alleles of these genes also have a Tex phenotype.--Several other E . coli mutations affecting related functions have been analyzed for their effects on Tn10 excision . Other mutations affecting the frequency of spontaneous mutations (mutT, polA, ung), different excision repair pathways (uvrA, uvrB) or the state of DNA methylation (dcm) have no effect on Tn10 excision . Mutations ssb-113 and mutD5, however, do increase Tn10 excision.--The products of the mismatch correction genes probably function in a coordinated way during DNA repair in vivo . Thus, mutations in these genes might also enhance transposon excision by a single general mechanism . Alternatively, since mutations in each gene have qualitatively and quantitatively different effects on transposon excision, defects in different mismatch repair genes may enhance excision by different mechanisms.

Bull Soc Pathol Exot Filiales, 1985, 78(5 Pt 2), 769 - 79
{Spiroplasmas and hematophagous arthropods; prospects in tropical pathology}; Chastel C et al.; Spiroplasmas are original wall-free prokaryotes which exhibit motility and a helical shape although lacking any cell wall or motile apparatus . They are known by phytopathologists because some of them are responsible for severe arthropod-borne plant diseases, such as Spiroplasma citri, the etiologic agent of "citrus stubborn disease" . Other spiroplasmas are pathogenic for a number of insects: drosophila, bees and beetles . More recently spiroplasmas were also isolated from ticks, mosquitoes, horse-flies and deer-flies opening a new chapter in medical pathology . These agents are of possible great significance in Tropical Medicine.

Membr Biochem, 1985, 5(4), 309 - 25
Resolution and reconstitution of H+ -ATPase complex from beef heart mitochondria; Joshi S et al.; Mitochondrial H+ -ATPase complex, purified by the lysolecithin extraction procedure, has been resolved into a "membrane" (NaBr-F0) and a "soluble" fraction by treatment with 3.5 M sodium bromide . The NaBr-F0 fraction is completely devoid of beta, delta, and epsilon subunits of the F, ATPase and largely devoid of alpha and gamma subunits of F1, where F0 is used to denote the membrane fraction and F1, coupling factor 1 . This is confirmed by complete loss of ATPase and Pi-ATP exchange activities . The addition of F1 (400 micrograms X mg-1 F0) results in complete restoration of oligomycin sensitivity without any reduction in the F1-ATPase activity . Presumably, this is due to release of ATPase inhibitor protein from the F1-F0 complex consequent to sodium bromide extraction . Restoration of Pi-ATP exchange and H+ -pumping activities require coupling factor B in addition to F1-ATPase . The oligomycin-sensitive ATPase and 32Pi-ATP exchange activities in reconstituted F1-F0 have the same sensitivity to uncouplers and energy transfer inhibitors as in starting submitochondrial particles from the heavy layer of mitochondria and F1-F0 complex . The data suggest that the altered properties of NaBr-F0 observed in other laboratories are probably inherent to their F1-F0 preparations rather than to sodium bromide treatment itself . The H+ -ATPase (F1-F0) complex of all known prokaryotic (3, 8, 9, 10, 21, 32, 34) and eukaryotic (11, 26, 30, 33, 35-37) phosphorylating membranes contain two functionally and structurally distinct entities . The hydrophilic component F1, composed of five unlike subunits, shows ATPase activity that is cold labile as well as uncoupler- and oligomycin-insensitive . The membrane-bound hydrophobic component F0, having no energy-linked catalytic activity of its own, is indirectly assayed by its ability to regain oligomycin sensitive ATPase and Pi-ATP exchange activities on binding to F1-ATPase (33) . The purest preparations of bovine heart mitochondrial F0 show seven or eight major components in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate or SDS-PAGE (1, 2, 12, 14), ranging from 6 to 54 ku in molecular weight (12) . The precise structure and polypeptide composition of mitochondrial F0 is not known . The F0 preparations from bovine heart reported so far have been derived from H+ -ATPase preparations isolated in the presence of cholate and deoxycholate (11, 33, 36, 37).(ABSTRACT TRUNCATED AT 400 WORDS)

J Exp Pathol, 1985 Fall, 2(3), 135 - 48
The triple helix: a potential mechanism for gene regulation; Minton KW; Nature has provided the cell with an exquisite recognition mechanism in the ability of oligoribonucleotide polypyrimidine sequences to specifically bind to native duplex DNA polypurine X polypyrimidine stretches in a sequence specific fashion . This binding does not require DNA strand separation, since the oligoribonucleotide inserts into the DNA major grove where sequence-specific determinants are recognized . Thus far, there is no evidence in either prokaryotes or eukaryotes that this specificity plays a role in regulation of nucleic acid functions . Current understanding of prokaryotic transcription regulatory mechanisms is fairly detailed and appears to exclude this possibility . Our understanding of regulation of eukaryotic transcription is less advanced; possible sites for transcriptional inhibition by triple helix formation in eukaryotes include polypyrimidine stretches involved in termination of tRNA transcripts and premature termination of SV40 late mRNA transcripts.

J Biol Chem, 1984 Dec 25, 259(24), 15257 - 63
Mobile domains in ribosomes revealed by proton nuclear magnetic resonance; Cowgill CA et al.; Ribosomes and subunits from eukaryotic and prokaryotic sources were studied by high-resolution proton magnetic-resonance spectroscopy . If all ribosomal components are firmly bound within the particle, then only broad spectra would be expected . However, relatively sharp resonances were found both in ribosomal subunits and in 70 or 80 S ribosomes . The regions of these mobile protein domains have been partially assigned in Escherichia coli ribosomes . Large and small ribosomal subunits were treated to remove selectively proteins L7/12 and S1, respectively . Sharp proton magnetic resonance spectra were not observed for the stripped large subunit showing that proteins L7/12 comprise the flexible protein region and that there is little other flexibility in the stripped subunit . Complete removal of S1 from the small subunit greatly reduced but did not abolish the sharp protein resonance peaks, indicating that protein S1 contains a substantial flexible component but that other flexible components remain in the stripped small subunit . Evidence for generality of these features of ribosome organization is provided by similar studies on ribosomes from eukaryotic sources.

Science, 1984 Dec 21, 226(4681), 1386 - 92
Enzymatic approach to syntheses of unnatural beta-lactams; Wolfe S et al.; Four enzymes associated with the transformation of the peptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) into the beta-lactam antibiotic desacetylcephalosporin C have been isolated from the prokaryotic organism Streptomyces clavuligerus and immobilized . Appropriate choice of the cofactors allows continuous and quantitative conversion of the peptide into either penicillins or cephalosporins at room temperature . The overall process includes four oxidations, two ring closures, and one epimerization . In contrast, cell-free transformations with the eukaryotic organism Cephalosporium acremonium do not proceed beyond the oxidation level of penicillin . The amino acids of the natural peptide ACV can be altered by chemical means; several of the resulting peptides are converted into novel antibiotics by the enzymes of Streptomyces clavuligerus.

J Lipid Res, 1984 Dec 15, 25(13), 1501 - 7
Bacterial membranes and lipid packing theory; Goldfine H; Recent physical studies on the lipids of biological membranes have emphasized the potential instability of the lamellar phase of mixtures of lipids containing unsaturated species of phosphatidylethanolamine, plasmenylethanolamine, or monoglycosyldiacylglycerols, all of which are important constituents of the membranes of different groups of prokaryotes . The polar lipid compositions of bacteria are examined in terms of lipid packing theory . This survey reveals that gram-negative species with high proportions of unsaturated fatty acids (greater than 65%), often have phosphatidylcholine (PC), in addition to the more common phosphatidylethanolamine (PE), phosphatidylglycerol, and cardiolipin . Physical studies have shown that PC is capable of inducing the bilayer phase when added to unsaturated PE . Many bacteria that are rich in unsaturated fatty acids and contain PC, have intracytoplasmic membrane systems (ICM), and the potential role of bilayer instability in the formation of ICM is discussed . Two groups of bacteria that are either natural fatty acid auxotrophs or utilize exogenous fatty acids when endogenous synthesis is inhibited, Acholeplasma laidlawii and the butyric acid-producing clostridia, are capable of adjusting their lipid class compositions according to the degree of unsaturation of their lipid aliphatic chains . Lipid class composition is also affected by growth temperature in both groups of organisms, and by incorporation of cholesterol in A . laidlawii . As the content of cis-unsaturated fatty acids or temperature is increased, lipids that form an unstable lamellar phase at physiological temperatures are replaced with lipids that have larger effective polar head groups, and can therefore form more stable bilayers.

J Biol Chem, 1984 Dec 10, 259(23), 14843 - 8
Synthesis and utilization of 8-azidoguanosine 3'-phosphate 5'-{5'-32P}phosphate . Photoaffinity studies on cytosolic proteins of Escherichia coli; Owens JR et al.; A family of guanosine 3',5'-phosphorylated nucleotides have been postulated to have pleiotypic regulatory properties in prokaryotes during the stringent response . To study proteins which may interact with nucleotides of this homologous series, a photoactive analog of guanosine 3',5'-diphosphate has been synthesized . The analog, 8-azidoguanosine 3'-phosphate 5'-{5'-32P}phosphate, proved to be an effective photoaffinity probe for two nucleotide-binding proteins of Escherichia coli sonicates . It predominately photolabels two proteins with approximate molecular weights of 86,000 and 65,000 (p86 and p65, respectively) . The Kd for p65 was approximately 10 microM; that for p86 was not determined . The nucleotide-binding sites were characterized by photolabeling in the presence of various nucleotides . The nucleotides guanosine 3',5'-dipyrophosphate, guanosine 3'-monophosphate 5'-diphosphate, and GTP were most effective at decreasing photoincorporation into p86; guanosine 3'-diphosphate 5'-monophosphate was least effective, with guanosine 3',5'-diphosphate and GMP having an intermediate effect . ATP increased photolabeling of p86 . However, ATP was one of the best of the nucleotides studied at decreasing photolabeling of p65, although guanosine 3'-monophosphate 5'-diphosphate, guanosine 3',5'-diphosphate, and GMP appeared only slightly less effective . The relative lack of effectiveness of guanosine 3'-diphosphate 5'-monophosphate inhibiting photolabeling of either protein supports observations that this nucleotide does not have a regulatory role in E . coli . The results presented indicate that the 8-azidoguanosine analogs of this homologous series will prove to be effective probes for studying the protein-nucleotide interactions involved in the stringent response.

Science, 1984 Dec 7, 226(4679), 1211 - 3
Cloned mycoplasma ribosomal RNA genes for the detection of mycoplasma contamination in tissue cultures; Gobel UB et al.; A cloned fragment of the mycoplasma ribosomal RNA operon was used as a molecular probe for the detection of mycoplasmas in cell cultures . According to the conditions of hybridization, the probe can detect prokaryotes in general or mycoplasmas specifically.

Proc Natl Acad Sci U S A, 1984 Dec, 81(24), 7747 - 51
A separate editing exonuclease for DNA replication: the epsilon subunit of Escherichia coli DNA polymerase III holoenzyme; Scheuermann RH et al.; DNA polymerase III (polIII) holoenzyme of Escherichia coli has 3'----5' exonuclease ("editing") activity in addition to its polymerase activity, a property shared by other prokaryotic DNA polymerases . The polymerization activity is carried by the large alpha subunit, the product of the dnaE gene . Mutations affecting the fidelity of DNA replication in vivo and the activity of 3'----5' exonuclease assayed in vitro are found in the dnaQ gene, which specifies the epsilon subunit . To determine whether epsilon carries the 3'----5' exonuclease activity, we have used an overproduction protocol to purify epsilon separately from the other subunits of polIII holoenzyme . We find that epsilon has 3'----5' exonuclease activity indistinguishable from that of polIII core, the subassembly of polIII holoenzyme consisting of the alpha, epsilon, and theta subunits . We conclude that the editing and polymerization activities of polIII holoenzyme reside on distinct subunits, in contrast to DNA polymerase I of E . coli and DNA polymerase of phage T4 . This functional separation may provide for regulation of exonucleolytic editing independently of polymerization, allowing cellular control of replication fidelity.

Gene, 1984 Dec, 32(1-2), 141 - 50
CII-dependent activation of the pRE promoter of coliphage lambda fused to the Escherichia coli galK gene; Fien K et al.; Using a cloning vector designed for the study of prokaryotic promoters by fusion to the Escherichia coli galactokinase gene (galK), we have constructed a plasmid in which the lambda pRE promoter controls galactokinase expression . A galK- host containing this plasmid has a Gal- phenotype since transcription from pRE requires activation by the lambda CII protein . When CII protein is provided by a prophage, galactokinase is synthesized at a rate dependent on the concentration of CII protein . A second plasmid was constructed in which the pRE promoter from phage 21 controls galactokinase expression . Transcription of the galK gene in this plasmid requires the phage 21 CII protein . Using this system, we demonstrate that the lambda and 21 pRE promoters are highly selective for their corresponding CII proteins . However, a cross-reaction between 21 pRE and the lambda CII protein was observed . In addition, we transferred the pRE-galK fusion unit from the plasmid to a phage, and then to the host chromosome in single copy . Galactokinase expression in this single copy pRE-galK system is also dependent on CII protein, which may be provided from a multicopy plasmid . The high concentration of CII protein provided by the plasmid results in maximal expression of the pRE-galK transcription unit . In this second system low levels of CII activity from CII- mutants are amplified and can be readily detected.

J Biol Chem, 1984 Nov 25, 259(22), 14101 - 4
Effect of base sequence on in vitro protein-chain termination; Ganoza MC et al.; It has been proposed that the sequences surrounding nonsense codons determine the efficiency of protein-chain termination . To test this hypothesis, the termination factor, RF-1, was purified to near homogeneity and was used to examine the specificity of in vitro prokaryotic termination as a function of the nature and number of bases adjacent to UAA . Oligomers with different nucleotide sequences surrounding UAA were synthesized and their conformation was analyzed by NMR spectroscopy . The activity of these oligomers in RF-1-dependent termination was assayed by the release of analogues of peptides, N-acetyl or N-formyl-methionine, that were bound to ribosomes as N-acetyl or N-formyl-Met-tRNAfMet with either AUG or AUG covalently linked to another oligoribonucleotide . In the former case, a second oligomer was added to stimulate release . When added to the AUG-bound intermediate, UAAUAA was 5-fold less effective in stimulating release of N-acetyl-Met by RF-1 than were UAA, UAAN (where N is any base), UAAUGA, or UAAUAG . Oligomers AUGUAA, AUGUUAA, and AUG(U)mUAA18-25 (where m = 1-5) stimulated release by RF-1, whereas AUGCUA, AUGCUAA, and other control polymers were inactive . The data suggest that recognition of UAA depends, at least in part, on the nature of the bases surrounding UAA . A loosely stacked conformation of UAA in the short messengers favors termination, whereas nucleosides which encourage strong base stacking restrict release.

Biochemistry, 1984 Nov 20, 23(24), 5697 - 703
Effect of polyamines and basic proteins on cleavage of DNA by restriction endonucleases; Pingoud A et al.; We have investigated the effect of the polyamines spermine, spermidine, and putrescine and the prokaryotic histone-like proteins NS1 and NS2 on the restriction endonuclease EcoRI catalyzed cleavage of plasmid and bacteriophage DNAs . At low concentrations of spermine and spermidine, the rate of DNA cleavage by EcoRI is increased, while high concentrations of spermine as well as of spermidine are inhibitory . These phenomena are also observed with other restriction endonucleases . They are, therefore, probably due to the interaction of the polyamines with the DNA . Putrescine does not have such an effect within the concentration range investigated . Remarkably, low concentrations of spermine and spermidine very efficiently suppress EcoRI activity . An inhibition of the EcoRI-catalyzed cleavage of DNA is also observed with NS1 and NS2, an effect that can be mimicked with other basic proteins that interact with DNA . The results are discussed in terms of the mechanism of restriction in vivo.

Can J Biochem Cell Biol, 1984 Nov, 62(11), 1181 - 9
The isolation of surface array proteins from bacteria; Koval SF et al.; The methods used for the isolation of regularly structured (RS) surface array proteins of a range of prokaryotes are described . Most RS proteins can be selectively solubilized from envelope preparations with low concentrations of urea or guanidine hydrochloride . Sodium dodecyl sulfate - polyacrylamide gel electrophoresis analysis of the protein extracts shows that most RS arrays are composed of a single polypeptide that may contain carbohydrate . The molecular weight of the proteins varies from 41 000 to 200 000 . Possible reasons for the presence of more than one polypeptide in RS protein preparations are discussed, as well as the evidence for proteolytic degradation of some RS proteins during isolation . Structural features of the RS proteins are described and the importance of protein conformation to assembly of the arrays is indicated.

Gene, 1984 Nov, 31(1-3), 23 - 30
The Nicotiana chloroplast genome . IX . Identification of regions active as prokaryotic promoters in Escherichia coli; Kong XF et al.; The galK-expression plasmid vector system pKO1 has been used to clone Nicotiana chloroplast (ct) promoters that function in Escherichia coli . The randomly cloned promoter-containing restriction fragments have been located on the ct genome and originate both from those regions encoding ribosomal and transfer RNAs and from locations elsewhere on the ct genome . The results provide the first demonstration that sequences which function as prokaryotic promoters exist in the ct genome.

Arch Microbiol, 1984 Nov, 140(1), 91 - 5
Studies on 1-methyl adenine transfer RNA methyltransferase of Mycobacterium smegmatis; Brahmachari V et al.; The presence of 1-methyl adenine in transfer RNA is a feature that Mycobacterium smegmatis shares with only a few other prokaryotes . The enzyme 1-methyl adenine tRNA methyl transferase from this source has been purified and the preliminary results show the presence of two activity peaks with different substrate specificity.

Mol Biol (Mosk), 1984 Nov-Dec, 18(6), 1486 - 96
{Binding of the yeast phenylalanine tRNA with Escherichia coli ribosomes . Effect of the removal of a modified base from the 3'-end of the anticodon on codon-anticodon interaction}; Katunin VI et al.; Phe-tRNAPhe+Y and N-acetyl-Phe-tRNAPhe+Y from yeast interact with prokaryotic 30S subunits and 70S ribosomes with slightly lower affinity than respective tRNA's of E . coli (decrease of standard free energy change of interaction less than 10%) . The removal of Y-base from Phe-tRNAPhe+Y results in two orders of magnitude decrease of association constant of Phe-tRNAPh-Ye with P site of the 30S X poly(U) complex and one ordef of magnitude or more of that with A site . The same modification decreases the association constants of Phe-tRNAPhe-Y and N-acetyl-Phe-tRNAPhe-Y 60 and 15 times respectively with P site of the 70S X poly(U) complex . In the absence of poly(U) the affinity of N-acetyl-Phe-tRNAPhe-Y to P-site of 70S ribosome was 20-fold lower than that of native N-acetyl-Phe-tRNAPhe+Y . The sign of interaction enthalpy of N-acetyl-Phe-tRNAPhe+/-Y and Phe-tRNAPhe-Y changes below 6-7 degrees C exposing the hydrophobic part of P-site interactions . Similar removal of Y-base does not change both the enthalpy of interaction with P-site and magnesium concentration dependence.

Nucleic Acids Res, 1984 Oct 25, 12(20), 7771 - 85
Sequences of the coding and flanking regions of the large ribosomal subunit RNA gene of mosquito mitochondria; HsuChen CC et al.; We have sequenced a 1.6 kbp region of the mosquito (Aedes albopictus) mitochondrial genome containing the large ribosomal subunit ("LSU") RNA gene, and have located the ends of the gene by S1 protection analysis and by comparison with RNA sequences . The gene is preceded by a tRNAval gene and followed by genes for tRNAIeuUAG (rather than tRNAleuUAA, as in mammalian mitochondria) and an extended reading frame homologous to mammalian URF1 . It is approximately 1335 residues long and is very low (17%) in G + C . The 5' half is even lower in G + C (9%), and shows little apparent homology to other LSU RNA classes . The 3' half is relatively rich (26%) in G + C and has many stretches of homology to prokaryotic and mammalian mitochondrial LSU RNA.

J Biol Chem, 1984 Oct 25, 259(20), 12696 - 704
Expression of a mammalian fatty acid-binding protein in Escherichia coli; Lowe JB et al.; Rat liver fatty acid-binding protein (FABP) is a 14,200-Da polypeptide that is abundantly represented in the cytosol of liver and small intestinal epithelial cells . It may play an important role in the intracellular transport and metabolism of fatty acids . We have recently determined its sequence from an analysis of cloned cDNAs (Gordon, J.I., Alpers, D.H., Ockner, R . K., and Strauss, A.W . (1983) J . Biol . Chem . 258, 3356-3363) . With the availability of full-length cloned FABP cDNAs, genetic manipulation of the coding region introduced into an appropriate expression vector could provide a powerful tool for analyzing the molecular details of fatty acyl-protein interaction . We, therefore, have inserted the coding sequence of rat FABP cDNA into a prokaryotic expression vector . Synthetic oligodeoxynucleotides were used to "properly" space the initiator methionine residue downstream from a Shine-Dalgarno ribosome-binding site . FABP transcription in the chimeric plasmid was under the direction of the leftward promotor of phage lambda . Promotor activity in turn was regulated by a thermolabile repressor specified by a defective lambda prophage contained in the host chromosome . When grown at permissive temperatures, rat liver FABP represented approximately 0.8% of radiolabeled soluble bacterial proteins . Edman degradation of FABP purified from Escherichia coli lysates indicated that it was intact . However, its NH2 terminus was not acetylated as it is in mammalian tissues . A frameshift mutation was introduced in vitro into the coding region of FABP cDNA . The effects of this on fatty acyl-protein interaction were examined using a solid phase (oleic acid-Sepharose) binding assay . Amino acid sequence rearrangements in the COOH-terminal region of rat liver FABP had a significant effect on its ability to bind fatty acids as well as on its stability in bacteria.

Nucleic Acids Res, 1984 Oct 11, 12(19), 7479 - 502
A universal model for the secondary structure of 5.8S ribosomal RNA molecules, their contact sites with 28S ribosomal RNAs, and their prokaryotic equivalent; Vaughn JC et al.; The phylogenetic approach (ref . 1) has been utilized in construction of a universal 5.8S rRNA secondary structure model, in which about 65% of the residues exist in paired structures . Conserved nucleotides primarily occupy unpaired regions . Multiple compensating base changes are demonstrated to be present in each of the five postulated helices, thereby forming a major basis for their proof . The results of chemical and enzymatic probing of 5.8S rRNAs (ref . 13, 32) are fully consistent with, and support, our model . This model differs in several ways from recently proposed 5.8S rRNA models (ref . 3, 4), which are discussed . Each of the helices in our model has been extended to the corresponding bacterial, chloroplast and mitochondrial sequences, which are demonstrated to be positionally conserved by alignment with their eukaryotic counterparts . This extension is also made for the base paired 5.8S/28S contact points, and their prokaryotic and organelle counterparts . The demonstrated identity of secondary structure in these diverse molecules strongly suggests that they perform equivalent functions in prokaryotic and eukaryotic ribosomes.

Isr J Med Sci, 1984 Oct, 20(10), 1025 - 7
Mycoplasma-like organisms (MLO), pathogens of the plant yellows diseases, as a model of coevolution between prokaryotes, insects and plants; Caudwell A; There is no satisfactory theory to explain how parasites whose effect is to kill or to weaken their hosts have been able to survive evolution . A good explanation may be found in the study of mycoplasma-like organisms (MLO) . We show here the existence of natural cycles of MLO between symptomless wild plants and unaffected vectors . Such natural cycles may be the basis of a coevolutionary process by which the prokaryotes and the whole cycle have persisted to our time . Disease outbreaks occur with intrusion of a cultivated plant (direct mode) or an imported insect (indirect mode) . From an evolutionary point of view, the natural cycle constitutes a protection for the host plants and natural vectors against the introduction of foreign plants or vectors as competitors in their ecological niche . A role of MLO and other parasites in evolution could be to provide stability to ecosystems, which is necessary for new characters to emerge.

EMBO J, 1984 Oct, 3(10), 2315 - 8
Analysis of the distribution of charged residues in the N-terminal region of signal sequences: implications for protein export in prokaryotic and eukaryotic cells; von Heijne G; A statistical analysis of the distribution of charged residues in the N-terminal region of 39 prokaryotic and 134 eukaryotic signal sequences reveals a remarkable similarity between the two samples, both in terms of net charge and in terms of the position of charged residues within the N-terminal region, and suggests that the formyl group on Metf is not removed in prokaryotic signal sequences.

Radiat Res, 1984 Oct, 100(1), 205 - 10
An oxygen effect for gamma-radiation induction of radiation resistance in yeast; Mitchel RE et al.; Previous evidence in a lower eukaryote indicated the lack of an oxygen effect for ionizing radiation induction of radioresistance {P . E . Bryant, Nature (London) 261, 588-590 (1976)}, suggesting that the signal for induction may be different from that in prokaryotes, where DNA damage by a variety of agents has been shown to induce SOS . type repair and radioresistance . We show here that prior exposure to a sublethal dose of gamma radiation caused induction of radioresistance in Saccharomyces cerevisiae, and that the rate of induction was proportional to the initial dose . Irradiation in oxygen produced a higher rate of increase in resistance, per unit dose, than irradiation in nitrogen, with an oxygen enhancement ratio of 2.8 . The maximum level of resistance reached in response to saturating doses was the same regardless of the presence or absence of O2 during the initial irradiation . At less than saturating doses, however, the maximum increase in resistance was higher, per unit dose, following irradiation in the presence of O2 than in its absence . Oxic doses which gave the maximum possible in increase the level of resistance were considerably less than those required to produce the maximum rate of increase in resistance . These results suggest a difference in the level at which DNA damage saturates these processes . These results also indicate that the use of DNA damage as a signal for induction of radiation resistance has probably been conserved during the evolution of prokaryotes to lower eukaryotes.

FEBS Lett, 1984 Oct 1, 175(2), 203 - 7
Mechanism of protein biosynthesis in prokaryotic cells . Effect of initiation factor IF1 on the initial rate of 30 S initiation complex formation; Pon CL et al.; To define the step at which translational initiation factor IF1 exercises its stimulation, initial rate kinetic analyses of 30 S initiation complex formation were carried out in the presence and absence of this factor . It was shown that, without affecting the affinity of the ribosomes either for the initiator tRNA or for the poly(AUG) used as template, IF1 increases approximately 2.5-fold the limiting Vmax of the 'pre-ternary complex'----ternary complex transition which represents the rate-limiting step in 30 S initiation complex formation . This kinetic effect titrates with the 30 S ribosomal subunit which must therefore represent the target of IF1 action.

DNA, 1984 Oct, 3(5), 365 - 76
Interaction of small nuclear ribonucleoproteins with simian virus 40 in CV-1 cells: is U2 snRNA involved in regulating replication?
Savouret JF, Cathala G, Eberhardt NL, Miller WL, Baxter JD.
The small nuclear RNAs (snRNAs) in African Green Monkey kidney cells (CV-1 cells) were examined by polyacrylamide gel electrophoresis . Methodology was developed to improve their extraction from enriched fractions . Cellular fractionation studies and subsequent analysis of these RNAs indicate that they are tightly associated with chromatin . Treatment of cells with alpha-amanitin totally suppressed transcription of U1, U2, U4, U5, and partially suppressed transcription of U6, suggesting that these snRNAs are transcribed by RNA polymerase II . Upon infection of the cells by simian virus 40 (SV40), overall transcription of these and other cellular RNAs was stimulated . Gel filtration and formaldehyde crosslinking studies indicated that the ribonucleoproteins (snRNPs) containing snRNAs are associated with the viral minichromosome . Nucleotide sequence comparisons show extensive sequence complementarity between the 5' end of U2 RNA, the replication origin of SV40, and a prokaryotic RNA (RNA I) that is involved in control of plasmid replication . The clustered homologies between these RNAs and the association of snRNAs with the SV40 chromosome suggest that snRNAs may be evolutionarily related to small RNAs from plasmids and are consistent with an hypothesis that U2 RNA may be involved in DNA replication.

Gene, 1984 Oct, 30(1-3), 251 - 5
A versatile plasmid system for the study of prokaryotic transcription signals in Escherichia coli; de Boer HA; For comparing the relative efficiencies of Escherichia coli promoters, a modified plasmid system, pKO-2 and pKM-2, has been constructed using short synthetic DNA fragments . The new vectors were derived from the plasmids pKO-1 and pKM-1 . The plasmids contain seven clustered unique restriction sites which can be used for promoter insertions . Also, three adjacent stop codons were introduced to abort any undesired translational initiation from various upstream origins . The DNA sequence of any insert in pKO-2 and pKM-2 can be determined rapidly by the supercoiled plasmid DNA sequencing method using a single oligonucleotide primer . The plasmid pKM-2 is especially suitable for the cloning and sequence determination of strong promoters.

Gene, 1984 Oct, 30(1-3), 211 - 7
A new sensitive method for qualitative and quantitative assay of neomycin phosphotransferase in crude cell extracts; Reiss B et al.; A general method is described for the detection and quantification of low amounts of neomycin phosphotransferase in crude cell extracts . The assay is based on the electrophoretic separation of the enzyme from other interfering proteins and detection of its enzymatic activity by in situ phosphorylation of the antibiotic kanamycin . Both kanamycin and {gamma-32P}ATP acting as substrates are embedded in an agarose gel placed on the polyacrylamide gel containing the separated proteins . After the enzymatic reaction, the phosphorylated kanamycin is transferred to P81 phosphocellulose ion exchange paper and the radiolabeled kanamycin is visualised by autoradiography . With this method 1 ng of active enzyme can easily be detected . Both prokaryotic and eukaryotic cell extracts can be examined, and changes in the size of enzymatically active proteins can be determined.

Gene, 1984 Oct, 30(1-3), 147 - 56
Expression of prokaryotic genes for hygromycin B and G418 resistance as dominant-selection markers in mouse L cells; Santerre RF et al.; Novel bacterial resistance genes were cloned and expressed as dominant selection markers in mammalian cells . Escherichia coli genes coding for resistance to the aminocyclitol antibiotics hygromycin B (Hm) and G418 were cloned into the eukaryotic expression plasmid pSV5GPT {Mulligan and Berg, Proc . Natl . Acad . Sci . USA 78 (1981) 2072-2076} . Mouse cells normally sensitive to 100 micrograms/ml Hm were transformed with these plasmids and selected in 200 micrograms/ml Hm . Transformants resistant to as much as 1 mg/ml Hm and 500 micrograms/ml G418 were isolated . Cell extracts contained an acetyltransferase activity capable of acetylating G418 and an Hm aminocyclitol phosphotransferase activity . Plasmid DNA sequences were identified by Southern blot analysis of high Mr DNA isolated from transformed cells.

J Bacteriol, 1984 Oct, 160(1), 184 - 91
lon transcriptional regulation of genes necessary for capsular polysaccharide synthesis in Escherichia coli K-12; Trisler P et al.; It has previously been observed that Escherichia coli lon mutations increase the levels of enzymes involved in the synthesis of colanic acid capsular polysaccharide (A . Markovitz, p . 415-462, in I . Sutherland, ed., Surface Carbohydrates of the Prokaryotic Cell, 1977) . To determine how lon regulates these enzymes, we have isolated, mapped, and characterized lac operon and lac protein fusions to genes necessary for capsule synthesis by the Mu d(lac Amp) in vivo fusion technique of Casadaban and Cohen (M . J . Casadaban and S . N . Cohen, Proc . Natl . Acad . Sci . U.S.A . 76:4530-4533, 1979) . At least five genes have been identified which share a common pattern of regulation: they are transcribed at low levels in lon+ strains and at significantly higher levels in lon strains . These genes are located in a cluster close to udk at 45 min on the E . coli map; we have named these genes cpsA, B, C, D, and E . An additional locus, cpsF, located at 90 min, is regulated in a similar manner to cpsA to E but is not essential for colanic acid synthesis . Similar studies on the transcriptional regulation of fusions in the gal and manA operons, also necessary for colanic acid synthesis, do not show significant regulation by the lon locus . Therefore, the regulatory system described here does not extend to all genes in the colanic acid synthesis pathway.

J Mol Biol, 1984 Sep 25, 178(3), 509 - 31
Cloning and sequence analysis of the Escherichia coli 4.5 S RNA gene; Hsu LM et al.; The structure of the Escherichia coli gene coding for the metabolically stable 4.5 S RNA has been determined by cloning and DNA sequence analysis . Results from Southern hybridization assays carried out prior to cloning show the 4.5 S DNA to be limited to a single locus in the E . coli K12 genome . A 5.4 X 10(3) base DNA fragment containing the 4.5 S DNA was cloned into plasmid pBR322 for restriction, hybridization and sequence analyses . Cells harboring the cloned gene overproduce the 4.5 S RNA by 15-fold under normal culturing conditions; however, no effect on growth rate is observed . DNA sequencing revealed only one copy of the 4.5 S RNA gene, with a deduced RNA sequence both longer at 114 bases and slightly different from the RNA sequence reported earlier . A promoter structure immediately preceding the structural gene shows good agreement with the prokaryotic consensus sequence at both the -35 and -10 regions . In addition, a G + C-rich sequence between the Pribnow box and the start of transcription agrees well with an apparent consensus sequence found for other stable RNA genes also under stringent control . No clearly recognizable termination signal was found immediately downstream from the 3' terminus of the 4.5 S DNA, although structural elements with that potential appear to occur . A potential coding sequence for a protein occurs about 100 bases downstream from the 4.5 S DNA, suggesting the possibility of a dual function 4.5 S RNA-mRNA transcript.

J Mol Biol, 1984 Sep 15, 178(2), 389 - 414
Crystal structure at 2.6 A resolution of the complex of subtilisin BPN' with streptomyces subtilisin inhibitor; Hirono S et al.; The crystal structure of the complex of a bacterial alkaline serine proteinase, subtilisin BPN', with its proteinaceous inhibitor SSI (Streptomyces subtilisin inhibitor) was solved at 2.6 A resolution . Compared with other similar complexes involving serine proteinases of the trypsin family, the present structure is unique in several respects . (1) In addition to the usual antiparallel beta-sheet involving the P1, P2 and P3 residues of the inhibitor, the P4, P5 and P6 residues form an antiparallel beta-sheet with a previously unnoticed chain segment (residues 102 through 104, which was named the S4-6 site) of subtilisin BPN' . (2) The S4-6 site does not exist in serine proteinases of the trypsin family, whether of mammalian or microbial origin . (3) Global induced-fit movement seems to occur on SSI: a channel-like structure in SSI where hydrophobic side-chains are sandwiched between two lobes becomes about 2 A wider upon complexing with subtilisin . (4) The complex is most probably a Michaelis complex, as in most of the other complexes . (5) The main role of the "secondary contact region" of SSI seems to be to support the reactive site loop ("primary contact region") . Steric homology of the two contact regions between the inhibitors of the SSI family and the pancreatic secretory trypsin inhibitor-ovomucoid inhibitor family is so high that it seems to indicate divergent evolutionary processes and to support the general notion as to the relationship of prokaryotic and eukaryotic genes put forward by Doolittle (1978).

Biochemistry, 1984 Sep 11, 23(19), 4289 - 94
Kinetics of incorporation of O6-methyldeoxyguanosine monophosphate during in vitro DNA synthesis; Snow ET et al.; O6-Methyldeoxyguanosine triphosphate (m6dGTP), known to be produced in vivo by methylation of deoxyguanosine triphosphate with simple methylating mutagens, is utilized by prokaryotic DNA polymerases during in vitro replication of synthetic and natural DNA template-primers . A study of the kinetic behavior of m6dGTP during DNA replication in vitro and of its effect on DNA replication indicates that m6dGTP acts as an analogue of dATP with Kappm of about 6 microM for Escherichia coli DNA polymerase I (Klenow fragment) compared to the Kappm of about 0.8 microM for dATP . m6dGTP is not incorporated in the complete absence of dATP (a competitive inhibitor) . m6dGTP also inhibits in vitro DNA synthesis . Different DNA polymerases behave differently in utilization and turnover of m6dGTP . T4 DNA polymerase shows stronger discrimination against m6dGMP incorporation than either T5 DNA polymerase or E . coli DNA polymerase I . The possibility that m6dGTP is unlikely to contribute significantly to in vivo mutation is discussed.

Nature, 1984 Sep 27-Oct 3, 311(5984), 392 - 4
Genetic effects of injection of Rous sarcoma virus DNA into polar plasm of early Drosophila melanogaster embryos; Gazaryan KG et al.; Retroviral proviruses and the transposable elements of eukaryotic genomes are structurally similar . The biological significance of eukaryotic transposable elements has not been examined extensively but it is known that, like prokaryotic transposons, these elements can induce mutations in adjacent genes and cause their transposition . It is of interest to determine whether retroviral proviruses have the same mutagenic and gene transposing ability as transposable elements, particularly because the retrovirus genome is assumed to have originated from transposable elements of lower eukaryotes . The transfer of DNA sequences into animal zygotes or embryos by microinjection is a promising experimental approach for eluxidating their functions: when foreign DNAs were introduced into a mouse germ line, mutations were induced and at least in some mice, the mutation was caused by the insertion of a retroviral sequence . We have introduced Rous sarcoma virus (RSV) DNA into a germ line of Drosophila melanogaster, and describe here the resultant genetic effects.

Isr J Med Sci, 1984 Sep, 20(9), 765 - 7
Deletions, duplications and rearrangements in mycoplasma ribosomal RNA gene sequences; Neimark H; Mycoplasma genomes lack several entire ribosomal RNA gene sets, and the remaining RNA genes lack some nucleotides . Analysis of mycoplasma 5S RNAs utilizing generalized models for prokaryotic 5S RNA secondary structure allows us to specify the molecular locations and identify many of the nucleotides missing from mycoplasma 5S RNAs and to detect the presence of unusual structural features . The most extensive alterations occur in the 5S RNAs from M . mycoides subsp . capri and M . capricolum, which have a similar pattern . Each of these 5S RNAs, as well as the Spiroplasma BC3 5S RNA, has a concentration of deletions in helix V, and they also share several unusual structures . We also noted the occurrence in mycoplasma 5S RNAs of numerous repeated sequences . The genetic process of unequal crossing-over is suggested as the explanation for the repeated sequences observed in mycoplasma RNAs.

EMBO J, 1984 Sep, 3(9), 2021 - 8
Synthetic donor and acceptor splice sites function in an RNA polymerase B (II) transcription unit; Rautmann G et al.; We have synthesised a 32-bp oligonucleotide containing sequences conforming to the consensus sequences for donor and acceptor splice sites . The oligonucleotide has been inserted into an RNA polymerase B (II) transcription unit and the resulting recombinant used to study the splicing mechanism . Our findings are as follows: (i) the synthetic sites function when separated by several different prokaryotic or eukaryotic DNA fragments providing bulk intron sequence, (ii) intron size need not be greater than 29 bp, (iii) an AG dinucleotide 11 bp upstream from the invariant AG of an acceptor splice site renders the latter non-functional, and (iv) sequence changes distant from splice sites can affect the efficiency of their utilisation.

J Gen Microbiol, 1984 Sep, 130 ( Pt 9), 2325 - 38
The variable T model for gram-negative morphology; Koch AL et al.; Gram-negative micro-organisms possess only a very thin murein sacculus to resist the stress caused by the internal hydrostatic pressure . The sacculus consists of at most one molecular layer of peptidoglycan in an extended conformation . It must grow by the insertion and cross-linking of new murein to the old before the selective cleavages of the stress-bearing murein are made which allow wall enlargement . Since insertion of new murein occurs all over the surface of Escherichia coli (even in completed poles), the internal pressure would tend to force the cells into a spherical shape and prevent both cylindrical elongation and cell division . Of course, Gram-negative bacteria do achieve a variety of shapes and do divide . Because prokaryote cells, unlike eukaryotic cells, do not have cytoskeletons and contractile proteins to transduce biochemical free energy into the mechanical work needed to achieve aspherical shapes and to divide, this paradox seems to be resolvable only by postulating that the details of the biochemical mechanism for wall growth vary in different regions of the surface, affecting the work required to enlarge the wall locally . Depending on the degree and rate of change in the biochemical energetics, it is possible to account for rod and the other more complex shapes of Gram-negative bacteria . Division occurs in Gram-negative organisms by the development of constrictions that progressively invade the cytoplasm . The work to cause these morphological processes must ultimately derive from the biochemical process of the stress-bearing wall formation . A biophysical basis for cell division in these prokaryotic organisms is proposed.

Mol Biol (Mosk), 1984 Sep-Oct, 18(5), 1233 - 48
{Primary and spatial structure of tRNA}; Tukalo MA; The recent achievements in studying of structure of tRNA are considered in the present paper . A brief analysis of the new methods for sequencing tRNA was carried out . Due to the development of these methods about 300 tRNA primary structures have been determined . Comparison of the primary tRNA structures gives us the possibility to divide them into seven classes: prokaryotic initiator tRNAs and eukaryotic initiator tRNAs; prokaryotic elongator tRNAs and eukaryotic elongator tRNAs; archaebacterial tRNAs; and mitochondrial tRNAs of lower and higher eukaryotes . Structural properties of the tRNAs of each of these classes are discussed . The second part of the paper is devoted to the three-dimensional structure of tRNA . Recent data in this field obtained by X-ray crystallographic technique as well as by high-resolution NMR and chemical modification methods are reviewed.

EMBO J, 1984 Sep, 3(9), 2145 - 9
Nucleotide sequence of the prokaryotic mobile genetic element IS30; Dalrymple B et al.; The complete nucleotide sequence of the mobile genetic element IS30, a resident of Escherichia coli K12, is 1221 bp long . A large open reading frame, preceded by possible transcription and translation control signals, could encode a basic protein of 383 amino acids which might presumably function as transposase . No large in-frame open reading frame is present on the opposite strand . The 26 bp long terminal inverted repeats have some sequence homology with the c-end of the phage Mu genome and with the terminal inverted repeats of the Halobacterium halobium insertion element ISH50 . The IS30 sequence has no significant homology with any other sequenced prokaryotic insertion sequences.

Nucleic Acids Res, 1984 Aug 24, 12(16), 6427 - 42
Multiple enhancer domains in the 3' terminus of the Prague strain of Rous sarcoma virus; Laimins LA et al.; The 3' terminal region of the Prague strain of Rous sarcoma virus (PrRSV) contains at least three distinct domains that comprise two functional enhancer elements . Two of these domains (designated B and C) are found in the U3 region of the 3' long terminal repeat (LTR) while the third (designated A) is located in the sequences immediately preceding the LTR termed XSR sequences . Combinations of adjacent domains {e.g., (A + B or B + C)} are capable of activating the expression of the SV40 early promoter (21 bp repeats and TATA box) coupled to coding sequences from the prokaryotic gene chloramphenicol acetyltransferase (CAT) while a single domain is inactive . Furthermore, duplication or triplication of the central domain B restores activity . The related, Schmidt-Ruppin, strain of RSV, contains an almost identical 3' LTR element, but differs in the enhancer sequences immediately preceding the 3' LTR . A model is presented in which the sequence differences may contribute to the difference in disease spectrum of transformation defective (td) variants of these viruses.

Nucleic Acids Res, 1984 Aug 10, 12(15), 6197 - 220
Xenopus laevis 28S ribosomal RNA: a secondary structure model and its evolutionary and functional implications; Clark CG et al.; Based upon the three experimentally derived models of E . coli 23S rRNA (1-3) and the partial model for yeast 26S rRNA (4), which was deduced by homology to E . coli, we derived a secondary structure model for Xenopus laevis 28S rRNA . This is the first complete model presented for eukaryotic 28S rRNA . Compensatory base changes support the general validity of our model and offer help to resolve which of the three E . coli models is correct in regions where they are different from one another . Eukaryotic rDNA is longer than prokaryotic rDNA by virtue of introns, expansion segments and transcribed spacers, all of which are discussed relative to our secondary structure model . Comments are made on the evolutionary origins of these three categories and the processing fates of their transcripts . Functionally important sites on our 28S rRNA secondary structure model are suggested by analogy for ribosomal protein binding, the GTPase center, the peptidyl transferase center, and for rRNA interaction with tRNA and 5S RNA . We discuss how RNA-RNA interactions may play a vital role in translocation.

Nature, 1984 Aug 2-8, 310(5976), 376 - 81
3-A resolution structure of a protein with histone-like properties in prokaryotes; Tanaka I et al.; The 3-A structure of DNA-binding protein II, which exhibits histone-like properties in bacteria, has been determined . The molecule is dimeric and appears to bind to the phosphate backbone of DNA through two symmetry-related arms . A mechanism by which the protein induces DNA supercoiling is proposed.

Genetics, 1984 Aug, 107(4), 537 - 49
Expression of a DNA replication gene cluster in bacteriophage T4: genetic linkage and the control of gene product interactions; Gerald WL et al.; The results of this study bear on the relationship between genetic linkage and control of interactions between the protein products of different cistrons . In T4 bacteriophage, genes 45 and 44 encode essential components of the phage DNA replication multiprotein complex . T4 gene 45 maps directly upstream of gene 44 relative to the overall direction of reading of this region of the phage chromosome, but it is not known whether these two genes are cotranscribed . It has been shown that a nonsense lesion of T4 gene 45 exerts a cis-dominant inhibitory effect on growth of a missense mutant of gene 44 but not on growth of phage carrying the wild-type gene 44 allele . In previous work, we confirmed these observations on polarity of the gene 45 mutation but detected no polar effects by this lesion on synthesis of either mutant or wild-type gene 44 protein . In the present study, we demonstrate that mRNA for gene 44 protein is separable by gel electrophoresis from gene 45-protein-encoding mRNA . That is, the two proteins are not synthesized from one polycistronic message, and the cis-dominant inhibitory effect of the gene 45 mutation on gene 44 function is probably expressed at a posttranslational stage . We propose that close genetic linkage, whether or not it provides shared transcriptional and translational regulatory signals for certain clusters of functionally related cistrons, may determine the intracellular compartmentalization for synthesis of proteins encoded by these clusters . In prokaryotes, such linkage-dependent compartmentation may minimize the diffusion distances between gene products that are synthesized at low levels and are destined to interact.

Biochem Genet, 1984 Aug, 22(7-8), 749 - 67
Structural homology between Drosophila melanogaster and Escherichia coli acidic ribosomal proteins; Chooi WY et al.; Antibodies raised against Drosophila melanogaster ribosomal proteins (r-proteins) were used to examine possible structural relationships between eukaryotic and prokaryotic r-proteins . The antisera were raised against either groups of r-proteins or individually purified r-proteins . Two antisera showed a cross-reaction with total Escherichia coli r-proteins in Ouchterlony double immunodiffusion assays: an antiserum against the D . melanogaster small subunit protein S14 (anti-S14) and an antiserum against a group of D . melanogaster r-proteins (anti-TP80) . The specificity of the antisera and the identity of the homologous E . coli r-proteins were characterized by using immunooverlay and immunoblot assays . These assays indicated that anti-S14 was highly specific for protein S14 and anti-TP80 was a multispecific serum that recognized several of the D . melanogaster ribosomal proteins . The E . coli protein homologous to D . melanogaster protein S14 was identified as E . coli protein S6 . By adsorption of the anti-TP80 serum, we determined that D . melanogaster protein 7/8 is homologous to the acidic E . coli protein L7/L12 . D . melanogaster acidic protein 13 was also shown to be immunologically related to D . melanogaster protein 7/8.

Nucleic Acids Res, 1984 Jul 25, 12(14), 5639 - 46
The nucleotide sequences of the genes coding for tRNAArgUCU, tRNAArgACG and tRNAAsnGUU on Spirodela oligorhiza chloroplast DNA; Keus RJ et al.; The nucleotide sequences of the genes coding for tRNAArgUCU, tRNAArgACG and tRNAAsnGUU on chloroplast DNA of Spirodela oligorhiza have been determined . All three genes are expressed . 5' Proximal to these genes sequences are found homologous to prokaryotic promoter sequences, which might be involved as transcriptional start motifs.

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