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-m