J Bacteriol, 1994 Jan, 176(1), 198 - 205 Translation initiation factor IF1 is essential for cell viability in Escherichia coli; Cummings HS et al.; Translation initiation factor IF1 is a highly conserved element of the prokaryotic translational apparatus . It has been demonstrated earlier that the factor stimulates in vitro the initiation phase of protein synthesis . However, no mutation in its gene, infA, has been identified, and a role for IF1 in translation has not been demonstrated in vivo . To elucidate the function of IF1 and determine if the protein is essential for cell growth, the chromosomal copy of infA was disrupted . Cell viability is maintained only when infA is expressed in trans from a plasmid, thereby demonstrating that IF1 is essential for cell growth in Escherichia coli . Cells depleted of IF1 exhibit few polysomes, suggesting that IF1 functions in the initiation phase of protein synthesis. J Virol Methods, 1994 Jan, 46(1), 39 - 50 Prokaryotic expression of a large fragment of the most antigenic cytomegalovirus DNA-binding protein (ppUL44) and its reactivity with human antibodies; Ripalti A et al.; We isolated and characterized from a lambda gt11 expression library clones expressing portions of human cytomegalovirus (HCMV)-p52 . This nonstructural viral protein is encoded by UL44 and is known to be one of the best IgM reactive antigens . The reactivity of these clones was studied with human antibody and the gene fragment coding for the most immune-reactive portion of p52 (aa 202-434) was cloned in a prokaryotic expression vector, pROS, which overexpresses the antigen as a fusion protein to a truncated molecule of beta-galactosidase. Am J Trop Med Hyg, 1994, 50(4 Suppl), 20 - 6 Protein expression in yeast as an approach to production of recombinant malaria antigens; Bathurst IC; The selection of a system suitable for expression of recombinant malaria antigens for vaccine development is, in the final analysis, empirical . However, experience gained with both malaria antigens and other recombinant proteins has provided helpful guidelines . Recombinant DNA technology has been successfully applied to the development of vaccines against a number of human diseases . For example, recombinant DNA-derived hepatitis B virus surface antigen has been produced from both prokaryotic and eukaryotic systems . Yeast has been demonstrated to be an excellent host for the expression of recombinant proteins with uses in diagnostics, therapeutics, and vaccine production . Both intracellular and secretory systems have been developed and optimized for the production of high levels of recombinant proteins . Recombinant DNA technology, and in particular yeast expression systems, have been successfully used to produce malaria antigens, several of which have been protective in various animal models . In contrast, attempts to produce sufficient quantities of antigens for a malaria vaccine from in vitro cultures of the malaria parasite have been unsuccessful . Recombinant proteins can be produced and purified from yeast in large quantities and at low cost, each being requirements for a vaccine to be used in a global vaccination program against malaria. Mol Gen Genet, 1994 Jan, 242(2), 201 - 8 Functional organization of the riboflavin biosynthesis operon from Bacillus subtilis SHgw; Mironov VN et al.; We have sequenced 6006 bp DNA of a region from the Bacillus subtilis SHgw chromosome known to contain riboflavin biosynthesis genes (rib gene cluster, 210 degrees on the B . subtilis genetic map) . Five of the seven open reading frames found within the sequence are shown to represent the genes ribG, ribB, ribA, ribH and ribTD . The calculated molecular masses for the putative translation products are 39,305, 23,481, 44,121, 16,287 and 14,574 daltons respectively . The five rib genes are transcribed as a polycistronic 4277 nucleotide messenger RNA . The steady-state level of the transcript is negatively regulated by riboflavin . A cis-acting element necessary for regulation was mapped by analysis of constitutive mutations within the 5' untranslated region of the operon . The element is at least 48 bp in length and does not bear obvious similarity to well defined prokaryotic regulatory elements . The molecular mechanism of regulation remains unknown, but the data presented argue against regulation by attenuation. J Mol Evol, 1994 Jan, 38(1), 1 - 17 Molecular evolution of the HSP70 multigene family; Boorstein WR et al.; Eukaryotic genomes encode multiple 70-kDa heat-shock proteins (HSP70s) . The Saccharomyces cerevisiae HSP70 family is comprised of eight members . Here we present the nucleotide sequence of the SSA3 and SSB2 genes, completing the nucleotide sequence data for the yeast HSP70 family . We have analyzed these yeast sequences as well as 29 HSP70s from 24 additional eukaryotic and prokaryotic species . Comparison of the sequences demonstrates the extreme conservation of HSP70s; proteins from the most distantly related species share at least 45% identity and more than one-sixth of the amino acids are identical in the aligned region (567 amino acids) among all proteins analyzed . Phylogenetic trees constructed by two independent methods indicate that ancient molecular and cellular events have given rise to at least four monophyletic groups of eukaryotic HSP70 proteins . Each group of evolutionarily similar HSP70s shares a common intracellular localization and is presumed to be comprised of functional homologues; these include heat-shock proteins of the cytoplasm, endoplasmic reticulum, mitochondria, and chloroplasts . HSP70s localized in mitochondria and plastids are most similar to the DnaK HSP70 homologues in purple bacteria and cyanobacteria, respectively, which is consistent with the proposed prokaryotic origin of these organelles . The analyses indicate that the major eukaryotic HSP70 groups arose prior to the divergence of the earliest eukaryotes, roughly 2 billion years ago . In some cases, as exemplified by the SSA genes encoding the cytoplasmic HSP70s of S . cerevisiae, more recent duplication events have given rise to subfamilies within the major groups . The S . cerevisiae SSB proteins comprise a unique subfamily not identified in other species to date . This subfamily appears to have resulted from an ancient gene duplication that occurred at approximately the same time as the origin of the major eukaryotic HSP70 groups. Mol Microbiol, 1994 Jan, 11(1), 9 - 13 Mammalian and Escherichia coli signal recognition particles; Luirink J et al.; Recent evidence from both biochemical and genetic studies indicates that protein targeting to the prokaryotic cytoplasmic membrane and the eukaryotic endoplasmic reticulum membrane may have more in common than previously thought . A ribonucleoprotein particle was identified in Escherichia coli that consists of at least one protein (P48 or Ffh) and one RNA molecule (4.5S RNA), both of which exhibit strong sequence similarity with constituents of the mammalian signal recognition particle (SRP) . Like the mammalian SRP, the E . coli SRP binds specifically to the signal sequence of presecretory proteins . Depletion of either P48 or 4.5S RNA affects translation and results in the accumulation of precursors of several secreted proteins . This review discusses the recent studies and speculates on the position of the SRP in the complex network of protein interactions involved in translation and membrane targeting in E . coli. Mol Microbiol, 1994 Jan, 11(1), 203 - 18 Glycogen in Bacillus subtilis: molecular characterization of an operon encoding enzymes involved in glycogen biosynthesis and degradation; Kiel JA et al.; Although it has never been reported that Bacillus subtilis is capable of accumulating glycogen, we have isolated a region from the chromosome of B . subtilis containing a glycogen operon . The operon is located directly downstream from trnB, which maps at 275 degrees on the B . subtilis chromosome . It encodes five polypeptides with extensive similarity to enzymes involved in glycogen and starch metabolism in both prokaryotes and eukaryotes . The operon is presumably expressed by an E sigma E-controlled promoter, which was previously identified downstream from trnB . We have observed glycogen biosynthesis in B . subtilis exclusively on media containing carbon sources that allow efficient sporulation . Sporulation-independent synthesis of glycogen occurred after integration of an E sigma A controlled promoter upstream of the operon. Protein Eng, 1994 Jan, 7(1), 57 - 64 Evolutionary divergence and conservation of trypsin; Rypniewski WR et al.; The trypsin sequences currently available in the data banks have been collected and aligned using first the amino acid sequence homology and, subsequently, the superposed crystal structures of trypsins from the cow, the bacterium Streptomyces griseus and the fungus Fusarium oxysporum . The phylogenetic tree constructed according to this multiple alignment is consistent with a continuous evolutionary divergence of trypsin from a common ancestor of both prokaryotes and eukaryotes . Comparison of crystal structures reveals a strict conservation of secondary structure . Similarly, in the alignment of all the sequences, insertions and deletions occur only in regions corresponding to loops between the secondary structure elements in the known crystal structures . The conserved residues cluster around the active site . Almost all conserved residues can be associated with one of the basic functional features of the protein: zymogen activation, catalysis and substrate specificity . In contrast, the residues of the hydrophobic core of the protein and the calcium ion binding sites are generally not conserved . The conserved features of trypsin and the nature of the conservation are discussed in detail. Microbios, 1994, 78(317), 229 - 36 Gene expression in Frankia: characterization of promoters; Cournoyer B et al.; Promoter regions harbouring typical -10 and -35 sites of prokaryotes were isolated from the genome of various species of Frankia using an Escherichia coli promoter-probe plasmid . These promoter regions initiated transcription in E . coli and Streptomyces cells . The consensus promoter sequence for the -10-like site was T A G/A G G/A T and for the -35-like site was T T G T/A C G . Despite the GC pressure observed in Frankia, functionally important positions in the promoter are conserved . As a consequence of these studies, a marker gene having properties compatible with the transcriptional and translational processes of Frankia was built . It is an npt-II gene under the control of three copies of one of the Frankia-E . coli type promoters studied. Immunogenetics, 1994, 40(2), 129 - 34 Cloning and sequence analysis of candidate human natural killer-enhancing factor genes; Shau H et al.; A cytosol factor from human red blood cells enhances natural killer (NK) activity . This factor, termed NK-enhancing factor (NKEF), is a protein of 44,000 M(r) consisting of two subunits of equal size linked by disulfide bonds . NKEF is expressed in the NK-sensitive erythroleukemic cell line K562 . Using an antibody specific for NKEF as a probe for immunoblot screening, we isolated several clones from a lambda gt11 cDNA library of K562 . Additional subcloning and sequencing revealed that the candidate NKEF cDNAs fell into one of two catagories of closely related but non-identical genes, referred to as NKEF A and B . They are 88% identical in amino acid sequence and 71% identical in nucleotide sequence . Southern blot analysis suggests that there are two to three NKEF family members in the genome . Analysis of predicted amino acid sequences indicates that both NKEF A and B are cytosol proteins with several phosphorylation sites each, but that they have no glycosylation sites . They are significantly homologous to several other proteins from a wide variety of organisms ranging from prokaryotes to mammals, especially with regard to several well-conserved motifs within the amino acid sequences . The biological functions of these proteins in other species are mostly unknown, but some of them were reported to be induced by oxidative stress . Therefore, as well as for immunoregulation of NK activity, NKEF may be important for cells in coping with oxidative insults. Environ Mol Mutagen, 1994, 23(4), 294 - 8 Effects of spermine on formation of HGPRT- mutants induced by ethylmethanesulfonate, methylmethanesulfonate, and mitomycin C in V79 Chinese hamster cells; Fiorio R et al.; Spermine is a polyamine found in bacteria, animal, and plant tissues . It is involved in a variety of biological processes, and its interaction with DNA stabilizes the secondary structure of the double helix . Spermine is one of the first reported antimutagens, reducing the mutation rate in several prokaryotic test systems, while in eukaryotic organisms conflicting results have been obtained . In light of the significant antimutagenic effect of spermine, it is important to evaluate its activity in mammalian cells in culture . The present study was undertaken to evaluate the ability of spermine to suppress the level of HGPRT- mutants induced by ethylmethanesulfonate, methylmethanesulfonate, and mitomycin C . Spermine reduced the mutation frequency induced by ethylmethanesulfonate and methylmethanesulfonate but did not affect survival; with mitomycin C survival was reduced but mutation rate was not influenced. Exp Clin Endocrinol, 1994, 102(3), 262 - 8 Paracrine and autocrine functions of the placenta: a key to the success of viviparity? Heap RB. The evolution of specific nuclear transcriptional regulators has endowed tissues of the reproductive system with responsiveness to small hydrophobic compounds such as steroids . Steroids are widely distributed in Nature and their distribution in prokaryotes and eukaryotes has given rise to the concept that their hormonal role came about by target organ specialization and not by the evolution of steroids themselves . Specific nuclear receptors for progesterone in the uterus are prominent during the establishment and maintenance of pregnancy . Anti-progesterone antagonists which interfere with receptor-mediated DNA activation abrogate pregnancy and thus emphasize the functional importance of the pathways by which the effects of progesterone as an extracellular signal are transduced . Comparative studies show that progesterone itself can be ovarian or placental in origin . This seems to reflect the evolution of different mechanisms of endocrine function rather than any obvious selective advantage being associated with the source of hormone secretion . For this reason, the question of whether the endocrine function of the placenta is obligatory for the adoption of viviparity in mammals is far from certain, and should be considered as an evolutionary option rather than a sine qua non . Of growing importance is the idea that the interaction between trophoblast and endometrial cells controls the degree of invasiveness at implantation and immunoreactivity.(ABSTRACT TRUNCATED AT 250 WORDS) DNA Seq, 1994, 4(4), 243 - 8 Nucleotide and deduced amino acid sequence of human cysteinyl-tRNA synthetase; Cruzen ME et al.; We have determined the nucleotide sequence of a cDNA coding human cysteinyl-tRNA synthetase . The predicted protein sequence of 638 amino acids has substantial sequence similarity to the E . coli cysteinyl-tRNA synthetase in the nucleotide-binding fold region that constitutes the potential active site . The results are consistent with the hypothesis that evolution of the full complement of aminoacyl-tRNA synthetases occurred prior to the divergence of prokaryotes and eukaryotes. Bioelectromagnetics, 1994, 15(4), 283 - 91 Sinusoidal 60 Hz electromagnetic fields failed to induce changes in protein synthesis in Escherichia coli; Kropinski AM et al.; Escherichia coli JM83 {F- ara delta(lac-proAB) rpsL {phi 80d delta (lacZ)M15}} in midlog growth phase at 30 degrees C were exposed to 60 Hz sinusoidal magnetic field of 3 mT of nonuniform diverging flux, inducing a nonuniform electric field with a maximum intensity of 32 microV/cm using an inductor coil . Exposed and unexposed control cells were maintained at 30.8 +/- 0.1 degrees C and 30.5 +/- 0.1 degrees C, respectively . Quadruplicate samples of exposed and unexposed E . coli cells were simultaneously radiolabeled with 35S-L-methionine at 10 min intervals over 2 hr . Radiochemical incorporation into proteins was analyzed via liquid scintillation counting and by denaturing 12.5% polyacrylamide gel electrophoresis . The results showed that E . coli exposed to a 60 Hz magnetic field of 3 mT exhibited no qualitative or quantitative changes in protein synthesis compared to unexposed cells . Thus small prokaryotic cells (less than 2 microns x 0.5 micron) under constant-temperature conditions do not alter their protein synthesis following exposure to 60 Hz magnetic fields at levels at 3 mT. Annu Rev Biochem, 1994, 63, 527 - 70 Escherichia coli single-stranded DNA-binding protein: multiple DNA-binding modes and cooperativities; Lohman TM et al.; There are now several well-documented SSBs from both prokaryotes and eukaryotes that function in replication, recombination, and repair; however, no "consensus" view of their interactions with ssDNA has emerged . Although these proteins all bind preferentially and with high affinity to ssDNA, their modes of binding to ssDNA in vitro, including whether they bind with cooperativity, often differ dramatically . This point is most clear upon comparing the properties of the phage T4 gene 32 protein and the E . coli SSB protein . Depending on the solution conditions, Eco SSB can bind ssDNA in several different modes, which display quite different properties, including cooperativity . The wide range of interactions with ssDNA observed for Eco SSB is due principally to its tetrameric structure and the fact that each SSB protomer (subunit) can bind ssDNA . This reflects a major difference between Eco SSB and the T4 gene 32 protein, which binds DNA as a monomer and displays "unlimited" positive cooperativity in its binding to ssDNA . The Eco SSB tetramer can bind ssDNA with at least two different types of nearest-neighbor positive cooperativity ("limited" and "unlimited"), as well as negative cooperativity among the subunits within an individual tetramer . In fact, this latter property, which is dependent upon salt concentration and nucleotide base composition, is a major factor influencing whether ssDNA interacts with all four or only two SSB subunits, which in turn determines the type of intertetramer positive cooperativity . Hence, it is clear that the interactions of Eco SSB with ssDNA are quite different from those of T4 gene 32 protein, and the idea that all SSBs bind to ssDNA as does the T4 gene 32 protein must be amended . Although it is not yet known which of the Eco SSB-binding modes is functionally important in vivo, it is possible that some of the modes are used preferentially in different DNA metabolic processes . In any event, the vastly different properties of the Eco SSB-binding modes must be considered in studies of DNA replication, recombination, and repair in vitro . Since eukaryotic mitochondrial SSBs as well as SSBs encoded by prokaryotic conjugative plasmids are highly similar to Eco SSB, these proteins are likely to show similar complexities . However, based on their heterotrimeric subunit composition, the eukaryotic nuclear SSBs (RP-A proteins) are significantly different from either Eco SSB or T4 gene 32 proteins . Further subclassification of these proteins must await more detailed biochemical and biophysical studies. Dev Biol Stand, 1994, 83, 7 - 11 The origins and consequences of genetic instability in prokaryotes; Summers DK; The causes of genetic change include errors in replication and repair, transposition and recombination . Together, these processes generate a reservoir of genetic variants from which selection will amplify any changes which increase host fitness . Recombination, transposition and slippage during replication may all result in large-scale DNA rearrangements . Homologous and illegitimate recombination generate deletions, inversions, duplications and fusions . Intramolecular transposition can cause deletion or inversion of DNA sequences adjacent to the mobile element, while intermolecular movement may lead to fusion of donor and target replicons when transposition is replicative . Of crucial importance is how genetic variation affects the fitness of the host cell . For a neutral variant the probability of eventual fixation is small, but changes which increase host cell fitness pose a more serious problem because these cells will eventually dominate the culture . The most effective way to combat the proliferation of variant plasmid cloning vectors is to block the processes by which they arise . Cloning vectors should be "stress-tested" and DNA sequence analysis programs can be used to screen for sequence repetition which should be removed, as far as possible, from both vectors and cloned sequences . It is also important that vectors place modest metabolic demands on their hosts in order to minimise the potential increase in fitness associated with changes in plasmid structure. Rocz Panstw Zakl Hig, 1994, 45(1-2), 151 - 5 {Usefulness of the fruit fly for assessment of mutagenicity of benzene, acetaldehyde and formaldehyde}; Krogulski A; Among the contaminants of water, soil and air the number of mutagenic and carcinogenic substances is increasing . For the assessment of health risk connected with the simple and cheap methods are necessary which could detected and measure the mutagenicity of these substances . The widely used tests using prokaryotes give negative results in the tests of certain substances which are carcinogenic in mammals . In the case of benzene and acetaldehyde Ames test gives false negative results, and in the case of formaldehyde the results are equivocal . An advantage of fruit fly Drosophila melanogaster used for this purpose is that its cell structures, enzymes and metabolic processes are similar to those of mammals . For the demonstration of mutagenicity of benzene, acetaldehyde and formaldehyde the test of somatic mutation and recombination SMART was carried out in these flies . The results confirmed the usefulness of the SMART test for the demonstration of the mutagenicity of contaminants in the environment. Drugs Exp Clin Res, 1994, 20(5), 177 - 83 Synthesis and antitumour activities of tetracyclic quinolone antineoplastic agents; Chu DT et al.; DNA topoisomerases, found in all prokaryotic and eukaryotic cells, play a key role in controlling the topological state of DNA . They are involved in DNA replication, RNA transcription and recombination affecting cell proliferation . Quinolone antibacterial agents have been shown to be inhibitors of DNA gyrase, a bacterial topoisomerase II enzyme . The eukaryotic topoisomerase II is the target of various cytotoxic agents such as adriamycin and etoposide . Due to the mechanistic similarities and sequence homologies shared by both bacterial and mammalian DNA topoisomerase II, we initiated a screening programme to search for quinolones as antitumour agents and reported the identification of a new class of quinolone, quinobenoxazines, having excellent in vitro cytotoxic activity comparable to adriamycin . In the continuation of this research work, we synthesized a series of amino-substituted quinobenoxazines and found that some of them possess more potent in vitro cytotoxicity than the parent unsubstituted quinobenoxazines . The chemical synthesis as well as biological properties of these tetracyclic quinolones are described. Mycoses, 1994, 37 Suppl 1, 13 - 27 {Festival lecture . The position of microorganisms in the global phylogenetic system of three domains}; Kandler O; New insights into genealogic relations among organisms due to similarities of 16S rRNA sequences of more than 1000 representatives of the various groups led to a classification of the presently known living beings into three domains: Bacteria, Archaea (Archaebacteria) and Eucarya . All medical relevant prokaryotes belong to the domain Bacteria . The systematics of the eukaryote microorganisms (protista and fungi) and their numerous medically relevant representatives will be widely changed in the near future due to latest scientific findings on phylogenetic relations . The retrospective analysis of the 16S rRNA phylogenetic tree led to assuming a chemolithoautotrophic origin of life and an early splitting into three domains in an early precellular phase of evolution. Hum Mol Genet, 1994, 3 Spec No, 1487 - 95 DNA methylation and cancer; Laird PW et al.; Changes in the pattern of DNA methylation have been a consistent finding in cancer cells . The mostly descriptive nature of these studies and the fact that both hypo- and hypermethylation have been observed at various loci have made it difficult to assess whether these changes are causally involved in the transformation process or whether they reflect the altered physiology of rapidly dividing cancer cells . It is clear, however, that DNA methylation plays an important role in the generation of mutations in human tumors . The high incidence of C-to-T transitions found in the p53 tumor-suppressor gene is attributed to the spontaneous deamination of 5-methylcytosine residues . The multiple observations linking DNA methylation to cancer can be resolved in a model proposing that the high rate of mutation at CpG dinucleotides is due in part to methyltransferase-facilitated deamination . Support for a role of DNA methyltransferase as a mutator enzyme is provided by work with a prokaryotic DNA methyltransferase under S-adenosyl-methionine methyl-donor limiting conditions . Methyl-donor limiting conditions might arise in early stages of tumor development, leading to high rates of methyltransferase-mediated CpG mutagenesis, as seen in human tumors . Such a mechanism is consistent with the frequently reported methionine auxotrophy of cancer cells and with the tumorigenic effects of methyl-deficient diets . Methyl deficiency in tumor cells is also consistent with the commonly observed global hypomethylation of tumor cell DNA, despite normal or even high levels of DNA methyltransferase expression. Biochimie, 1994, 76(5), 428 - 39 Potential secondary structure at the translational start domain of eukaryotic and prokaryotic mRNAs; Ganoza MC et al.; In order to identify conserved potential secondary structures within translational start sites, mRNA sequences derived from different species were studied with programs able to depict such features . The potential secondary structure of 71 bases around the initiator AUG or AUGs in the coding sequences of 290 eukaryotic mRNAs was first examined and compared to 290 similarly analyzed regions derived from prokaryotic mRNA sequences (Nucleic Acids Res (1987) 15, 345-360) . In both sets of sequences the initiator codon was often found to be in an open potential structure whereas a denser region characterized by nearly-periodic spacings defined the coding regions . Randomization of the sequences obliterated the observed patterns suggesting that the structure of the mRNA may determine these differences . Three sets of eukaryotic and prokaryotic mRNAs of approximately equal length were analyzed and found to preserve an open unpaired non-coding region 5' to the start codon . The start codon was found free of potential secondary structure in over 80% of all the sequences analyzed . These data, and study of mutants that restrict the accessibility of the start codon to the ribosomal initiation complex, suggest that both the prokaryotic and eukaryotic mRNA start sites must occur free of potential secondary structure for efficient initiation . A striking difference of the eukaryotic mRNA sequences analyzed was the high propensity of the coding region vicinal to the start codon to form secondary structures . Certain translation-defective mutants exhibit impaired formation of these secondary structures suggesting that the structure of the coding regions adjacent to the start codons of eukaryotic mRNAs may be an important, thus far unexamined, determinant of initiation . We propose that, for all genes studied, the transition in secondary structure between the coding and non-coding regions may be an important determinant of initiation. Antonie Van Leeuwenhoek, 1994, 65(4), 311 - 29 Protein structure, electron transfer and evolution of prokaryotic photosynthetic reaction centers; Blankenship RE; Photosynthetic reaction centers from a variety of organisms have been isolated and characterized . The groups of prokaryotic photosynthetic organisms include the purple bacteria, the filamentous green bacteria, the green sulfur bacteria and the heliobacteria as anoxygenic representatives as well as the cyanobacteria and prochlorophytes as oxygenic representatives . This review focuses on structural and functional comparisons of the various groups of photosynthetic reaction centers and considers possible evolutionary scenarios to explain the diversity of existing photosynthetic organisms. Annu Rev Microbiol, 1994, 48, 713 - 42 Antisense RNA control in bacteria, phages, and plasmids; Wagner EG et al.; Antisense RNA control is now recognized as an efficient and specific means of regulating gene expression at the posttranscriptional level . Almost all naturally occurring cases have been found in prokaryotes, often in their accessory genetic elements . Several antisense RNA systems are now well-understood, and these display a spectrum of mechanisms of action, binding pathways, and kinetics . This review summarizes antisense RNA control in prokaryotes, emphasizing the biology of the systems involved. Genetica, 1994, 93(1-3), 5 - 12 Transposable elements and adaptation of host bacteria; Blot M; A transposable element (TE) is a mobile sequence present in the genome of an organism . TEs can cause lethal mutations by inserting into essential genes, promoting deletions or leaving short sequences upon excision . They therefore may be gradually eliminated from mixed populations of haploid micro-organisms such as Escherichia coli if they cannot balance this mutation load . Horizontal transmission between cells is known to occur and promote the transfer of TEs, but at rates often too low to compensate for the burden to their hosts . Therefore, alternative mechanisms should be found by these elements to earn their keep in the cells . Several theories have been suggested to explain their long-term maintenance in prokaryotic genomes, but little molecular evidence has been experimentally obtained . In this paper, the permanence of transposable elements in bacterial populations is discussed in terms of costs or benefits for the element and for the host . It is observed that, in all studies yet reported, the elements do not behave in their host as selfish DNA but as a co-operative component for the evolution of the couple. Tsitologiia, 1994, 36(2), 131 - 47 {Oncogenesis in transgenic mice}; Shvemberger IN et al.; Oncogenesis in transgenic mice is at present a model, most adequately reflecting the natural conditions of tumor development . One of more important traits of this model is that it allows to study malignant growth simultaneously at all the structure-function levels in the context of the whole organism . This paper is a review of results of a series of experiments in which the localization of tumors was dependent or independent on the tissue specificity of a promoter, as well as development of multiple tumors with the use of viral regulatory sequences in genetic constructions . It has been shown that although a transgene is expressed in most of the tissues, tumors develop in some particular tissues only . These observations are interpreted by some authors in favour of the concept of multistep cancerogenesis . In this view, of primary importance are the results of studies on oncogenesis in transgenic mice, which contradict this concept and are regarded by their authors as an evidence of the possibility of a one-step transformation of normal cell into malignant one . The analysis of the obtained material enabled us to put forward an assumption that the key role in oncogenesis is played not only by certain genetic disturbances, but also by multi-level homeostatic mechanisms . Apparently, it is just the transgenic mice with cellular or viral oncogenes in their genome that represent a more adequate model for the detection of certain molecular-biological mechanisms underlying these disturbances . Also, of much importance is abundant material accumulated by now on oncogenesis of transgenic mice which shows a possibility of the effective use of various genetic constructions with prokaryotic and eukaryotic regulatory sequences, a possibility to induce not only tumors of some particular tissues, but also multiple hyperplastic and neoplastic changes in one and the same mouse . Development of tumors in such transgenic mice can be regarded as a model of different types of cancer disease. Biochimie, 1994, 76(10-11), 992 - 1004 HU and functional analogs in eukaryotes promote Hin invertasome assembly; Paull TT et al.; The prokaryotic protein HU functions as an accessory factor in many different biochemical reactions . We have characterized the role of HU in assembling the invertasome, an intermediate nucleoprotein complex involved in Hin-mediated site-specific recombination . Formation of this complex requires the looping of intervening DNA segments between sites bound by the Hin recombinase and the Fis protein . HU stimulates this process on substrates containing intervening segments of length < 100 bp . Characterization of the activity of HU in Hin-mediated recombination in vitro and in vivo yields evidence that its role in this reaction is primarily to facilitate the looping of the intervening DNA segment . By using this reaction as an assay, we identify proteins from mammals, yeast, trypanosomes, and wheat which can fulfill the same function in vitro . Using ligase-mediated circularization of short DNA fragments we also show that HU, the high mobility group (HMG) 1 and 2 proteins from mammals, and a protein from yeast can bend DNA extremely efficiently . These results support the view that this ubiquitous class of proteins enhance the assembly of nucleoprotein complexes under conditions of limited DNA flexibility. Antonie Van Leeuwenhoek, 1994, 66(1-3), 165 - 85 Metabolism of sulfate-reducing prokaryotes; Hansen TA; Dissimilatory sulfate reduction is carried out by a heterogeneous group of bacteria and archaea that occur in environments with temperatures up to 105 degrees C . As a group together they have the capacity to metabolize a wide variety of compounds ranging from hydrogen via typical organic fermentation products to hexadecane, toluene, and several types of substituted aromatics . Without exception all sulfate reducers activate sulfate to APS; the natural electron donor(s) for the ensuing APS reductase reaction is not known . The same is true for the reduction of the product bisulfite; in addition there is still some uncertainty as to whether the pathway to sulfide is a direct six-electron reduction of bisulfite or whether it involves trithionate and thiosulfate as intermediates . The study of the degradation pathways of organic substrates by sulfate-reducing prokaryotes has led to the discovery of novel non-cyclic pathways for the oxidation of the acetyl moiety of acetyl-CoA to CO2 . The most detailed knowledge is available on the metabolism of Desulfovibrio strains, both on the pathways and enzymes involved in substrate degradation and on electron transfer components and terminal reductases . Problems encountered in elucidating the flow of reducing equivalents and energy transduction are the cytoplasmic localization of the terminal reductases and uncertainties about the electron donors for the reactions catalyzed by these enzymes . New developments in the study of the metabolism of sulfate-reducing bacteria and archaea are reviewed. Acta Microbiol Immunol Hung, 1994, 41(3), 265 - 71 Blue-green pigmented symbionts of echiurids from polar marine environments; Reichardt W; Burrowing echiurid worms from marine sediments including sulfidic habitats of both the Antarctic Ocean and the Norwegian Sea were partly colonized by blue-green prokaryotic symbionts . These resembled free-living Prochlorophytes and contained 2-vinylpheophorbide-a5 as the only pigment being detectable by HPLC . The potential biogeochemical significance of these epizoic symbionts is discussed. Cold Spring Harb Symp Quant Biol, 1994, 59, 195 - 206 Regulation of the cryptic sequence-specific DNA-binding function of p53 by protein kinases; Hupp TR et al.; p53 is an allosterically regulated protein with a latent DNA-binding activity . Posttranslational modification of a carboxy-terminal regulatory site in vitro, by casein kinase II and protein kinase C, can activate the sequence-specific DNA-binding function of the wild-type protein . The latent form of p53 is produced in a variety of eukaryotic and prokaryotic cell lines, including E . coli, Sf9 insect cells, and C6 cells, indicating that the activation of p53 in vivo is rate-limiting . In addition, phosphorylation of p53 at the protein kinase C site and activation in vivo correlate with the loss of reactivity of active p53 protein to the carboxy-terminal antibody, PAb421 . These results suggest that two highly conserved protein kinases modify polypeptide structure through a common biochemical mechanism and that different enzymatic pathways may channel information into the carboxy-terminal regulatory site of p53, allosterically activating its function as a tumor suppressor. DNA Res, 1994, 1(3), 103 - 14 Cloning and characterization of the ribosomal protein genes in the spc operon of a prokaryotic endosymbiont of the pea aphid, Acyrthosiphon kondoi; Abe R et al.; To correlate a prokaryotic endosymbiont in the pea aphid, Acyrthosiphon kondoi, with the endosymbionts in related aphid species as well as with free-living bacteria and subcellular organelles, and to study the mode of its gene expression within aphid cells, we have cloned and characterized the genes encoding ribosomal proteins S3, L16, L29, S17, L14, L24, L5, S14, S8, L6, L18, S5, L30, L15 and secretion protein Y (Sec Y) from the S10 and spc ribosomal protein gene operons of this endosymbiont . The organization of these genes is identical to that in Escherichia coli, and their nucleotide sequences are highly similar (87% identity) to the corresponding E . coli genes . They are much less similar to the corresponding chloroplast and mitochondrial genes . The guanine plus cytosine G+C content of the genes of the A . kondoi endosymbiont is much higher than those of the endosymbionts in related aphid species reported so far . It appears either that the A . kondoi endosymbiont is derived from an ancestral bacterium different from those in other aphids or that its G+C content increased in a relatively short time after the evolutionary divergence of its host. Genetica, 1994, 94(2-3), 157 - 72 Control of mRNA processing and decay in prokaryotes; Alifano P et al.; Post-transcriptional mechanisms operate in regulation of gene expression in bacteria, the amount of a given gene product being also dependent on the inactivation rate of its own message . Moreover, segmental differences in mRNA stability of polycistronic transcripts may be responsible for differential expression of genes clustered in operons . Given the absence of 5' to 3' exoribonucleolytic activities in prokaryotes, both endoribonucleases and 3' to 5' exoribonucleases are involved in chemical decay of mRNA . As the 3' to 5' exoribonucleolytic activities are readily blocked by stem-loop structures which are usual at the 3' ends of bacterial messages, the rate of decay is primarily determined by the rate of the first endonucleolytic cleavage within the transcripts, after which the resulting mRNA intermediates are degraded by the 3' to 5' exoribonucleases . Consequently, the stability of a given transcript is determined by the accessibility of suitable target sites to endonucleolytic activities . A considerable number of bacterial messages decay with a net 5' to 3' directionality . Two different alternative models have been proposed to explain such a finding, the first invoking the presence of functional coupling between degradation and the movement of the ribosomes along the transcripts, the second one implying the existence of a 5' to 3' processive '5' binding nuclease' . The different systems by which these two current models of mRNA decay have been tested will be presented with particular emphasis on polycistronic transcripts. Int J Cancer Suppl, 1994, 8, 64 - 9 Epitope mapping of SCLC-cluster-2 MAbs and generation of antibodies directed against new EGP-2 epitopes; Helfrich W et al.; Western blot analysis proved that all cluster-2 MAbs recognize identical or overlapping disulfide-bond-dependent epitopes, indicating the presence of a disulfide-bond-stabilized EGP-2 domain carrying highly immunodominant non-linear epitopes . The apparent immunodominance of this domain makes it difficult to generate and select antibodies against other potentially useful EGP-2 epitopes . Using PCR, we have generated mutant EGP-2 cDNA (delta EGP-2) from which the coding sequences for a putative immunodominant 6-kDa intra-chain loop structure has been removed . delta EGP-2 transfected COS-7 cells reacted with MM104, an antibody detecting a linear epitope on EGP-2, but were not recognized by any cluster-2 MAb . To generate new anti-EGP-2 antibodies we constructed another mutant EGP-2 protein (delta EGP-2) from which additional domains, irrelevant for antibody generation (signal peptide, trans-membrane and cytoplasmic domains), were removed . delta EGP-2 was introduced in a prokaryotic expression system that adds a polyhistidine affinity tag to the delta EGP-2 N-terminus, making possible one-step purification by immobilized metal-ion-affinity chromatography (IMAC) . Western blot analysis showed that sera derived from mice immunized with purified delta EGP-2 had high-titer antibodies to reduced EGP-2 samples . We conclude that the availability of prokaryotic and eukaryotic EGP-2-expression constructs might facilitate the selection of new anti-EGP-2 MAbs otherwise difficult to obtain. Appl Environ Microbiol, 1994 Jan, 60(1), 45 - 50 A bioassay based on recombinant DNA technology for determining selenium concentration; Reches M et al.; The trace element selenium has recently attracted attention, particularly because (i) selenocysteine is involved in the active site of various prokaryotic and eukaryotic enzymes, some of which have a role in human health; (ii) selenocysteine incorporation into these proteins is coded by UGA codons; and (iii) as a result, selenocysteine is now considered to be the 21st amino acid in an expanded genetic code . Here, we built recombinant DNA constructs in which expression of the lac'Z gene is driven in Escherichia coli by UGA-directed selenocysteine incorporation . In this system, levels of beta-galactosidase activity are proportionally and specifically related to the presence and concentrations of several specific simple selenium derivatives . The system can thus be used as a sensitive bioassay for their determination . This bioassay is one of a few using recombinant DNA technology to provide a reporter for simple detection of a chemical trace element. J Mol Biol, 1993 Dec 20, 234(4), 1284 - 9 Molecular cloning of a P-type ATPase gene from the cyanobacterium Synechocystis sp . PCC 6803 . Homology to eukaryotic Ca(2+)-ATPases; Geisler M et al.; With oligonucleotide primers derived from P-type ATPase genes of different sources, a part of Synechocystis sp . PCC 6803 genomic DNA was amplified and used as hybridization probe for the Synechocystis gene . A 4.7 kb HindIII fragment was cloned and sequenced; it contains the open reading frame of the E1E2-ATPase . The Synechocystis ATPase (named PMA1) consists of 915 amino acids with a M(r) of 98,902; it has ten putative transmembrane domains and contains the conserved regions a to j common to all P-type ATPases . Its amino acid sequence shows less than 20% identity to prokaryotic ATPases but about 30% identity to eukaryotic Ca(2+)-ATPases . An alignment to rat kidney and yeast Ca(2+)-ATPase protein sequences shows homology in stalk regions and transmembrane domains domains which are thought to be involved in calcium binding and transport; these three ATPases reveal very similar hydropathy plots and form a separate group in the phylogenetic tree of P-type ATPases . The results strongly support the assumption that PMA1 of Synechocystis is a calcium translocating ATPase, possibly involved in regulatory processes with calcium as second messenger. Eur J Biochem, 1993 Dec 15, 218(3), 985 - 95 Overproduction, purification and characterization of the Escherichia coli ferritin; Hudson AJ et al.; Recent studies have indicated that Escherichia coli possesses at least two iron-storage proteins, the haem-containing bacterioferritin and ferritin . The ferritin protein has been amplified 600-fold to 11-14% of total cell protein in a bfr mutant and purified to homogeneity with an overall yield of 13% . The cellular ferritin content remained relatively constant throughout the growth cycle and amplification was accompanied by a 2.5-fold increase in cellular iron content . The isolated ferritin contained 5-20 non-haem iron atoms/holomer and resembled the eukaryotic ferritins rather than the prokaryotic bacterioferritins in containing no haem . The 24 subunits of this ferritin (M(r) 19,400) assemble into a spherical protein shell (12 +/- 1 nm diameter, M(r) 465,000) which sequesters at least 2000 iron atoms in vitro to form an electron-dense iron core of 7.9 +/- 1 nm diameter . Electron-microscopic and Mossbauer spectroscopic studies with iron-loaded ferritin showed that the core can be either crystalline (ferrihydrite) or amorphous, depending on the absence or presence of phosphate, respectively . Mossbauer spectroscopy with intact E . coli revealed a novel-high spin Fe(II) component which is enhanced in bacteria amplified for ferritin but not in the parental strain . Western blotting showed that ferritin and bacterioferritin are immunologically distinct proteins . E . coli is thus an organism containing both a ferritin and a bacterioferritin and the relative roles of the two iron-storage proteins are discussed in this study. Gene, 1993 Dec 15, 135(1-2), 49 - 56 Evolution of prokaryotic genomes; Arber W; Molecular genetics, which has its roots mainly in the development of microbial genetics in the middle of this century, not only greatly facilitates investigations of essential cellular functions, but also offers a means to better understand evolutionary progress . Spontaneous mutagenesis, the driving force of biological evolution, depends on a multitude of mechanistically distinct processes, many of which are already quite well understood . Often, enzymes act as variation generators, and natural gene vectors help to spread functional domains, entire genes and groups of genes across natural isolation barriers . In this overview, particular attention is given to comparing three selected natural strategies for the generation of genetic diversity: nucleotide substitution, DNA rearrangements, and gene acquisition . All of these mechanisms, as well as many others, appear to fulfill their specific roles in microbial evolution . Rather than being the result of an accumulation of errors, biological evolution may depend on a multitude of specific biological functions, as well as on a certain degree of intrinsic structural flexibility of biological molecules. Gene, 1993 Dec 15, 135(1-2), 175 - 82 Retroviral DNA integration: lessons for transposon shuffling; Skalka AM; Phylogenetic comparisons of retroviral IN (integrase) protein sequences have revealed homologies that extend to the retrotransposon and bacterial transposase families and have provided evidence for at least two functional domains . The N-terminal region is characterized by a Zn-finger-like array which is conserved in retrotransposons . The central region is defined by a D,D(35)E amino acid constellation which is conserved through the retrotransposons and several bacterial IS element transposases . Mutagenesis studies and biochemical analysis of the isolated central D,D(35)E domain support our original suggestion that this region contains the catalytic center of these proteins which must all share a similar enzymatic mechanism . These, and other recent findings suggest unexpected relationships between diverse pathways of nucleic acid metabolism in eukaryotes and prokaryotes. EMBO J, 1993 Dec 15, 12(13), 5019 - 27 Specific interaction of IHF with RIBs, a class of bacterial repetitive DNA elements located at the 3' end of transcription units; Boccard F et al.; The prokaryotic REP (repetitive extragenic palindromes) or PU (palindromic units) sequences are often associated with other repetitive elements, forming arrangements which have been called 'BIMEs' (bacterial interspersed mosaic elements) . It is estimated that the Escherichia coli chromosome carries approximately 300-500 BIMEs, whose biological role is at present unknown . We have identified a family of BIMEs consisting of two converging REP sequences flanking a 35 bp conserved segment which carries a static DNA bend and a binding site for IHF, the integration host factor of E.coli . We estimate that the E.coli genome harbors approximately 100 copies of this module, which we name 'RIB' (reiterative ihf BIME) . We have analyzed by gel retardation and by footprinting the in vitro interaction of IHF with individual RIBs, and shown that the protein binds strongly and specifically to their center . We have also demonstrated binding of IHF to the chromosomal population of RIBs, using a new approach which combines two-dimensional bandshift and Southern blotting . RIB elements are at the end of transcription units, and thus define a new class of ihf sites . Possible implications for genome structure and DNA topology are discussed. J Biol Chem, 1993 Dec 15, 268(35), 26602 - 6 Primary structure of pyruvate dehydrogenase kinase establishes a new family of eukaryotic protein kinases; Popov KM et al.; We recently reported molecular cloning of the branched chain alpha-ketoacid dehydrogenase kinase, the first mitochondrial protein kinase to be cloned (Popov, K . M., Zhao, Y., Shimomura, Y., Kuntz, M . J., and Harris, R . A . (1992) J . Biol . Chem . 267, 13127-13130) . From a search for proteins related to the branched chain alpha-ketoacid dehydrogenase kinase, a cDNA encoding the 434 amino acid residues corresponding to pyruvate dehydrogenase kinase has been cloned from a rat heart cDNA library . Evidence that the clone codes for pyruvate dehydrogenase kinase includes: (a) the deduced amino acid sequence is identical to the partial sequence of the kinase determined by direct sequencing; (b) expression of the cDNA in Escherichia coli resulted in synthesis of a protein that phosphorylated and inactivated the pyruvate dehydrogenase complex; (c) kinase activity of the recombinant protein is sensitive to inhibition by a specific inhibitor of pyruvate dehydrogenase kinase; and (d) antiserum raised against the recombinant protein recognized the protein subunit known to correspond to pyruvate dehydrogenase kinase in a highly purified preparation of the pyruvate dehydrogenase complex . Like the branched chain alpha-ketoacid dehydrogenase kinase, pyruvate dehydrogenase kinase lacks motifs usually associated with eukaryotic Ser/Thr-protein kinases . Considerable sequence similarity exists between these mitochondrial protein kinases and members of the prokaryotic histidine kinase family, a diverse set of sensing and response systems important in the regulation of bacterial processes . Thus, molecular cloning of these proteins establishes a new eukaryotic family of protein kinases that is related to a prokaryotic family of protein kinases. Eur J Biochem, 1993 Dec 15, 218(3), 963 - 71 The pur8 gene from the pur cluster of Streptomyces alboniger encodes a highly hydrophobic polypeptide which confers resistance to puromycin; Tercero JA et al.; A novel puromycin-resistance determinant (pur8) was isolated from one end of the pur cluster that encodes the puromycin biosynthetic pathway from Streptomyces alboniger and expressed in Streptomyces lividans . The gene pur8 induced antibiotic resistance that was highly specific for puromycin . The nucleotide sequence of pur8 contains an open reading frame of 1512 bp whose deduced amino acid sequence encodes a polypeptide (Pur8) with 14 possible transmembrane-spanning segments . It shows significant similarities to other known or putative transmembrane proteins, including a number which confer drug resistance in a variety of antibiotic-producing Streptomyces, Gram-positive and Gram-negative bacteria, and some solute transporters of prokaryotic and eukaryotic origin . As is probably the case for most of these proteins, Pur8 may be involved in active puromycin efflux energized by a proton-dependent electrochemical gradient . In addition, it could be implicated in secreting N-acetylpuromycin, the last intermediate of the biosynthesis pathway, to the environment. Nat Genet, 1993 Dec, 5(4), 327 - 37 The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene; Bull PC et al.; Wilson disease (WD) is an autosomal recessive disorder of copper transport, resulting in copper accumulation and toxicity to the liver and brain . The gene (WD) has been mapped to chromosome 13 q14.3 . On yeast artificial chromosomes from this region we have identified a sequence, similar to that coding for the proposed copper binding regions of the putative ATPase gene (MNK) defective in Menkes disease . We show that this sequence forms part of a P-type ATPase gene (referred to here as Wc1) that is very similar to MNK, with six putative metal binding regions similar to those found in prokaryotic heavy metal transporters . The gene, expressed in liver and kidney, lies within a 300 kb region likely to include the WD locus . Two WD patients were found to be homozygous for a seven base deletion within the coding region of Wc1 . Wc1 is proposed as the gene for WD. FEMS Microbiol Lett, 1993 Dec 1, 114(2), 121 - 8 Streptomyces lividans as host for heterologous protein production; Anne J et al.; Streptomycetes are Gram-positive soil bacteria with a well differentiated morphology . They are considered interesting candidates for the production of heterologous proteins for several reasons, including their efficient secretion mechanism by which the secreted proteins are localized into the culture supernatant . In view of this potential, this review article describes different aspects of gene expression and regulation in Streptomyces, and summarizes and discusses results obtained using Streptomyces lividans as host for secretion of heterologous proteins of prokaryotic and eukaryotic origin. J Cell Biol, 1993 Dec, 123(6 Pt 2), 1635 - 48 SMC1: an essential yeast gene encoding a putative head-rod-tail protein is required for nuclear division and defines a new ubiquitous protein family; Strunnikov AV et al.; The smc1-1 mutant was identified initially as a mutant of Saccharomyces cerevisiae that had an elevated rate of minichromosome nondisjunction . We have cloned the wild-type SMC1 gene . The sequence of the SMC1 gene predicts that its product (Smc1p) is a 141-kD protein, and antibodies against Smc1 protein detect a protein with mobility of 165 kD . Analysis of the primary and putative secondary structure of Smc1p suggests that it contains two central coiled-coil regions flanked by an amino-terminal nucleoside triphosphate (NTP)-binding head and a conserved carboxy-terminal tail . These analyses also indicate that Smc1p is an evolutionary conserved protein and is a member of a new family of proteins ubiquitous among prokaryotes and eukaryotes . The SMC1 gene is essential for viability . Several phenotypic characteristics of the mutant alleles of smc1 gene indicate that its product is involved in some aspects of nuclear metabolism, most likely in chromosome segregation . The smc1-1 and smc1-2 mutants have a dramatic increase in mitotic loss of a chromosome fragment and chromosome III, respectively, but have no increase in mitotic recombination . Depletion of SMC1 function in the ts mutant, smc1-2, causes a dramatic mitosis-related lethality . Smc1p-depleted cells have a defect in nuclear division as evidenced by the absence of anaphase cells . This phenotype of the smc1-2 mutant is not RAD9 dependent . Based upon the facts that Smc1p is a member of a ubiquitous family, and it is essential for yeast nuclear division, we propose that Smc1p and Smc1p-like proteins function in a fundamental aspect of prokaryotic and eukaryotic cell division. Genes Dev, 1993 Dec, 7(12B), 2629 - 40 Isolation and characterization of an Escherichia coli mutant defective in resuming growth after starvation; Siegele DA et al.; To understand the mechanisms that allow the enteric bacterium Escherichia coli to make the transitions between growth and stationary phase and to maintain cell viability during starvation, we have looked for mutants defective in stationary-phase survival (a Sur- phenotype) . In this paper we describe a conditional E . coli mutant, surB1, that grows normally and remains viable during stationary phase but is unable to exit stationary phase and resume aerobic growth at high temperature . Thus, the surB gene product is not required for cell survival per se but, rather, it is required for starved cells to reinitiate growth under restrictive conditions . Once growth has started, SurB function is no longer required . Mutant cells sense and respond to fresh medium but appear to arrest growth before the first cell division . The surB gene was mapped to 19.5 min on the E . coli chromosome, cloned, and sequenced . The surB gene product is predicted to be an integral membrane protein with multiple membrane-spanning regions and is homologous to the ATP-binding cassette (ABC) family of transporters, a large family of transport proteins found in both prokaryotic and eukaryotic cells . An open reading frame, designated ybjA, was found immediately upstream of surB and may be in an operon with surB . The predicted ybjA gene product is also homologous to the ABC transporter family and SurB and YbjA may function together in a common transport pathway . Either surB or ybjA may be the same gene as cydC, a gene described previously whose function is needed for the production of functional cytochrome d oxidase complexes . Consistent with this prediction, surB1 mutant cells were found to lack functional cytochrome d oxidase . However, the SurB- phenotype is not simply attributable to the absence of cytochrome d oxidase . Thus, the surB gene product may have an additional role in the cell. Eur J Biochem, 1993 Dec 1, 218(2), 311 - 20 Human aldehyde dehydrogenase . cDNA cloning and primary structure of the enzyme that catalyzes dehydrogenation of 4-aminobutyraldehyde; Kurys G et al.; Human liver aldehyde dehydrogenase (E3 isozyme), with wide substrate specificity and low Km for 4-aminobutyraldehyde, was only recently characterized {Kurys, G., Ambroziak, W . & Pietruszko, R . (1989) J . Biol . Chem . 264, 4715-4721} and in this study we report on its primary structure . Polyclonal antibodies, specific for the E3 isozyme and three oligonucleotide probes derived from amino acid sequence of the E3 protein, were used for isolation of the first cDNA clone encoding the human enzyme (1503 bp; coding for 440 amino acid residues) . Additional clones were obtained by using the first isolated clone as a probe . The largest clone of 1635 bp coded for 462 amino acid residues; it was longer at the 3'end of the cDNA non-coding region . The identity of the clone was established by DNA sequencing and by comparison with peptide sequences derived from the E3 protein, which constituted approximately 29% of the total primary structure of the E3 isozyme . The start codon was never encountered despite a variety of different approaches (500 amino acid residues were expected on the basis of SDS-gel molecular-mass determination of the E3 isozyme subunit) . Despite the great catalytic similarity between the E3 and E1 isozymes {Ambroziak, W . & Pietruszko, R . (1991) J . Biol . Chem . 266, 13011-13018}, the primary structure of the E3 isozyme has only approximately 40.6% of positional identity with that of the E1 isozyme . Sequence comparison with GenBank and Protein Identification Resource database sequences indicated no primary structure of aldehyde dehydrogenase more closely resembling the E3 isozyme than that of Escherichia coli betaine aldehyde dehydrogenase (52.7% positional identity), a prokaryotic enzyme specific for betaine aldehyde. Genes Dev, 1993 Dec, 7(12A), 2446 - 55 Amino-terminal amino acids modulate sigma-factor DNA-binding activity; Dombroski AJ et al.; Prokaryotic transcription initiation factor sigma is required for sequence-specific promoter recognition by RNA polymerase . Genetic studies have indicated that sigma itself interacts with DNA at the -10 and -35 promoter consensus sequences . Binding of Escherichia coli sigma 70 to DNA in vitro, however, can only be observed for truncated polypeptides lacking the amino-terminal amino acids . We have investigated the role of the amino terminus of E . coli sigma 70 in controlling DNA-binding ability . Deletion analysis indicates that amino acids within amino-terminal region 1.1 of sigma 70 inhibit DNA binding by the carboxy-terminal DNA-binding domains . Furthermore, inhibition of binding by the amino-terminal inhibitory domain of sigma 70 can be observed in trans . Likewise, the amino-terminal extensions of two alternative sigma-factors, E . coli sigma 32 and Bacillus subtilis sigma K, negatively affect the DNA binding activity of their carboxy-terminal domains . We propose that initiation of transcription is subject to modulation as a result of the composition and/or structure of the amino terminus of the sigma-subunit and that the sigma family of proteins belong to a larger class of intramolecularly regulated transcriptional effectors. Genes Dev, 1993 Dec, 7(12A), 2405 - 17 A splicing enhancer in the human fibronectin alternate ED1 exon interacts with SR proteins and stimulates U2 snRNP binding; Lavigueur A et al.; The inclusion of the 270-nucleotide human fibronectin ED1 exon in HeLa cells requires the presence of a centrally located 81-nucleotide exon sequence . We have conducted a series of in vitro experiments aimed at understanding the structural and functional features associated with this splicing enhancer (SE) . Using hybrid model pre-mRNA substrates, we show that the SE element markedly stimulates the use of the 3' splice site of ED1 . Deletion and replacement analysis identifies the stimulating sequences as a purine-rich stretch of 9 nucleotides (GAAGAAGAC) . The SE element stimulates splicing to the ED1 3' splice site from various positions within the exon except when placed beyond 293 nucleotides downstream from that 3' splice site . The action of the enhancer is not limited to the ED1 acceptor site because the SE element stimulates human beta-globin splicing and also induces the use of a 3' splice site in a prokaryotic sequence in vitro . We have explored the mechanism of action of the fibronectin splicing enhancer and found that the SE element is required for efficient assembly of early splicing complexes, allowing a more efficient interaction of the U2 snRNP with branch site sequences . In competition experiments, an RNA containing mainly SE sequences specifically abolished the action of the SE element, suggesting that factors bind the enhancer element to mediate stimulation of splicing . Using RNA mobility shift assays we show that SR proteins interact specifically with the SE element . Our results demonstrate that exon sequences lying in the SE element play a crucial role in specifying splice site recognition through interactions with factors binding to the 3' splice site. Mol Biol Cell, 1993 Dec, 4(12), 1239 - 50 Determination of the functional domains involved in nucleolar targeting of nucleolin; Creancier L et al.; Nucleolin (713 aa), a major nucleolar protein, presents two structural domains: a N-terminus implicated in interaction with chromatin and a C-terminus containing four RNA-binding domains (RRMs) and a glycine/arginine-rich domain mainly involved in pre-rRNA packaging . Furthermore, nucleolin was shown to shuttle between cytoplasm and nucleolus . To get an insight on the nature of nuclear and nucleolar localization signals, a set of nucleolin deletion mutants in fusion with the prokaryotic chloramphenicol acetyltransferase (CAT) were constructed, and the resulting chimeric proteins were recognized by anti-CAT antibodies . First, a nuclear location signal bipartite and composed of two short basic stretches separated by eleven residues was characterized . Deletion of either motifs renders the protein cytoplasmic . Second, by deleting one or more domains implicated in nucleolin association either with DNA, RNA, or proteins, we demonstrated that nucleolar accumulation requires, in addition to the nuclear localization sequence, at least two of the five RRMs in presence or absence of N-terminus . However, in presence of only one RRM the N-terminus allowed a partial targeting of the chimeric protein to the nucleolus. J Bioenerg Biomembr, 1993 Dec, 25(6), 647 - 69 Na+/H+ antiporters, molecular devices that couple the Na+ and H+ circulation in cells; Padan E et al.; Na+/H+ antiporters are universal devices involved in the Na+ and H+ circulation of both eukaryotes and prokaryotes, thus playing an essential role in the pH and Na+ homeostasis of cells . This review focuses on the major impact of the application of molecular biology tools in the study of the antiporters . These tools permit the verification of the role of the antiporters and provide insights into their unique biology . A novel signal transduction to Na+ involving nhaR, a positive regulator, controls the expression of nhaA in E . coli . A "pH sensor" regulates the activity of Na+/H+ antiporters, both in eukaryotes and prokaryotes . A most intricate signal transduction to pH involving phosphorylation steps controls the activity of nhel in higher mammals . The identification of Histidine 226 in the "pH sensor" of NhaA is a step forward towards the understanding of the pH regulation of these proteins. Curr Opin Cell Biol, 1993 Dec, 5(6), 971 - 6 Protein splicing: selfish genes invade cellular proteins; Neff NF; Protein splicing is a series of enzymatic events involving intramolecular protein breakage, rejoining and intron homing, in which introns are able to promote the recombinative transposition of their own coding sequences . Eukaryotic and prokaryotic spliced proteins have conserved similar gene structure, but little amino acid identity . The genes coding for these spliced proteins contain internal in-frame introns that encode polypeptides that apparently self-excise from the resulting host protein sequences . Excision of the 'protein intron' is coupled with joining of the two flanking protein regions encoded by exons of the host gene . Some introns of this type encode DNA endonucleases, related to Group I RNA intron gene products, that stimulate gene conversion and self-transmission. J Gen Microbiol, 1993 Dec, 139 ( Pt 12), 3185 - 95 Sequencing and analysis of the divergon comprising gtaB, the structural gene of UDP-glucose pyrophosphorylase of Bacillus subtilis 168; Soldo B et al.; Nucleotide sequencing revealed that gtaB, the structural gene of UDP-glucose pyrophosphorylase (EC 2.7.7.9), is part of a divergon-like genetic entity . The latter consists of two monocistronic operons gtaB and orfX, transcribed from a 245 bp regulatory region, each encoding an acidic protein with a molecular mass of 33.0 and 42.6 kDa, respectively . gtaB is transcribed from a distal PA promoter, and a proximal PB promoter which is negatively controlled by the Sin protein . Sin-mediated transcriptional attenuation and enhancement of PB and PD, respectively, suggest that these promoters control functions which antagonize each other . Transcription of orfX is mediated by a PA promoter . The regulatory region comprises four ATGAAA hexamers, present as two inverse repeats . Protein GtaB exhibits high homology to analogous prokaryotic enzymes, while OrfX shows 55.4% homology with the product of Escherichia coli o389, which is part of a regulatory unit involved in sugar processing . Mutations gtaB515 and gtaBg100, which define different bacteriophage adsorption patterns, were sequenced . They are transitions leading to substitution of amino acids which occupy conserved positions, and are thus likely to be part of an enzyme active site . The nature of the possible receptors for defective bacteriophages PBSY and PBSZ is discussed. Curr Opin Genet Dev, 1993 Dec, 3(6), 884 - 90 Origin and evolution of organelle genomes; Gray MW; Molecular data (particularly sequence analyses) have established that two eukaryotic organelles, the mitochondrion and the plastid, are the descendants of endosymbiotic (eu)bacteria whose closest living relatives are the alpha-Proteobacteria (mitochondrion) and Cyanobacteria (plastid) . This review describes recent data that favor the view that each organelle arose via this primary endosymbiotic pathway only once (monophyletic origin), such as the discovery of group I introns that appear to be structurally homologous and have identical insertion sites in metaphyte, chlorophyte and fungal mitochondrial genomes . However, it is also evident that the plastids in certain algal groups were acquired secondarily through a eukaryotic rather than a prokaryotic endosymbiont. Curr Opin Genet Dev, 1993 Dec, 3(6), 837 - 44 Genome organization in prokaryotes; Campbell AM; Most of the well-characterized prokaryotic genomes consist of double-stranded DNA organized as a single circular chromosome 0.6-10 Mb in length and one or more circular plasmid species of 2 kb-1.7 Mb . The past few years, however, have revealed some major variations in genome organization . In addition, a recent accumulation of data has shown that the location and orientation of the genes and repeated sequences (including prophages and transposons) on and among these elements is not always random . Some of the non-randomness is probably the result of unique historical events; in other cases it reflects selection for the optimization of function. Trends Biochem Sci, 1993 Dec, 18(12), 471 - 5 Control of gene activity in higher eukaryotic cells by prokaryotic regulatory elements; Gossen M et al.; Regulated gene expression systems for the study of gene function in prokaryotes and yeast have been available for several years . However, comparable systems in higher organisms are more complex and problematic . Recently, regulatory proteins from distant species have been used to establish highly specific control systems in eukaryotic cells . This is possible because their action can be modulated by effectors that are inert to the physiology of the organism or cell and therefore do not elicit pleiotropic effects . Such monospecific regulatory circuits open up new possibilities for the study of gene function in vivo. Proteins, 1993 Dec, 17(4), 363 - 74 Hundreds of ankyrin-like repeats in functionally diverse proteins: mobile modules that cross phyla horizontally? Bork P. Based on pattern searches and systematic database screening, almost 650 different ankyrin-like (ANK) repeats from nearly all phyla have been identified; more than 150 of them are reported here for the first time . Their presence in functionally diverse proteins such as enzymes, toxins, and transcription factors strongly suggests domain shuffling, but their occurrence in prokaryotes and yeast excludes exon shuffling . The spreading mechanism remains unknown, but in at least three cases horizontal gene transfer appears to be involved . ANK repeats occur in at least four consecutive copies . The terminal repeats are more variable in sequence . One feature of the internal repeats is a predicted central hydrophobic alpha-helix, which is likely to interact with other repeats . The functions of the ankyrin-like repeats are compatible with a role in protein-protein interactions. Mol Microbiol, 1993 Dec, 10(5), 917 - 22 Linear plasmids and chromosomes in bacteria; Hinnebusch J et al.; Linear plasmids and chromosomes were unknown in prokaryotes until recently but have now been found in spirochaetes, Gram-positive bacteria, and Gram-negative bacteria . Two structural types of bacterial linear DNA have been characterized . Linear plasmids of the spirochaete Borrelia have a covalently closed hairpin loop at each end and linear plasmids of the Gram-positive filamentous Streptomyces have a covalently attached protein at each end . Replicons with similar structures are more frequent in eukaryotic cells than in prokaryotes . Linear genomic structures are probably more common in bacteria than previously recognized, however, and some replicons may interconvert between circular and linear isomers . The molecular biology of these widely dispersed elements provides clues to explain the origin of linear DNA in bacteria, including evidence for genetic exchange between prokaryotes and eukaryotes. Mol Microbiol, 1993 Dec, 10(5), 903 - 9 In a class of its own--the RNA polymerase sigma factor sigma 54 (sigma N); Merrick MJ; Bacteria synthesize a number of different sigma factors which allow the co-ordinate expression of groups of genes owing to the ability of sigma to confer promoter-specific transcription initiation on RNA polymerase . In nearly all cases these sigmas belong to a single family of proteins which appear to be related structurally and functionally to the major Escherichia coli sigma factor, sigma 70 . A clear exception is the sigma factor sigma 54 (sigma N), encoded by rpoN, which represents a second family of sigmas that is widely distributed in prokaryotes . Studies of sigma 54 (sigma N) have demonstrated that this sigma is quite distinct both structurally and functionally from the sigma 70 family and the mode of transcription initiation which it mediates may have more in common with that found in eukaryotes than that which occurs with sigma 70 and its relatives. Proc Natl Acad Sci U S A, 1993 Dec 1, 90(23), 10967 - 71 Identification and functional analysis of chaperonin 10, the groES homolog from yeast mitochondria; Rospert S et al.; Chaperonin 60 (cpn60) and chaperonin 10 (cpn10) constitute the chaperonin system in prokaryotes, mitochondria, and chloroplasts . In Escherichia coli, these two chaperonins are also termed groEL and groES . We have used a functional assay to identify the groES homolog cpn10 in yeast mitochondria . When dimeric ribulose-1,5-bisphosphate carboxylase (Rubisco) is denatured and allowed to bind to yeast cpn60, subsequent refolding of Rubisco is strictly dependent upon yeast cpn10 . The heterologous combination of cpn60 from E . coli plus yeast cpn10 is also functional . In contrast, yeast cpn60 plus E . coli cpn10 do not support refolding of Rubisco . In the presence of MgATP, yeast cpn60 and yeast cpn10 form a stable complex that can be isolated by gel filtration and that facilitates refolding of denatured Rubisco . Although the potassium-dependent ATPase activity of E . coli cpn60 can be inhibited by cpn10 from either E . coli or yeast, neither of these cpn10s inhibits the ATPase activity of yeast cpn60 . Amino acid sequencing of yeast cpn10 reveals substantial similarity to the corresponding cpn10 proteins from rat mitochondria and prokaryotes. Infect Immun, 1993 Dec, 61(12), 5123 - 8 Differential protein expression and surface presentation generate high-frequency antigenic variation in Mycoplasma fermentans; Theiss PM et al.; Mycoplasma fermentans, a wall-less prokaryote, is currently under investigation as a potential human pathogen . Recently, several surface lipoproteins have been shown to vary in expression between M . fermentans strains . Using specific antibodies to these lipoproteins, we investigated the extent and nature of antigenic variation within this species . Immunoscreening of type strain PG18 agar-grown colonies revealed marked heterogeneity in expression of distinct surface lipoproteins . Subsequent isolation and propagation of clonal isolates established isogenic lineages which displayed high-frequency (10(-2) to 10(-5) per generation) antigenic phase variation . {35S}cysteine-labeled protein profiles and Western immunoblots of phase-variant clones showed that several distinct integral membrane proteins undergo noncoordinate variation in expression . In addition to differential expression of epitope-bearing lipoproteins, differential accessibility of epitopes to antibodies was also documented as a mechanism generating surface phenotypic variation . Examination of one strain-variant antigen showed high-frequency phase variation to underlie previously observed antigenic differences between strains of this species . Thus, M . fermentans has a complex system capable of creating rapid changes in surface mosaics . This may profoundly affect mycoplasma-host interactions and may limit the methods by which populations of M . fermentans may be studied in vivo. J Bioenerg Biomembr, 1993 Dec, 25(6), 613 - 20 Tinkering with transporters: periplasmic binding protein-dependent maltose transport in E . coli; Shuman HA et al.; Periplasmic binding protein-dependent transport systems represent a common mechanism for nutrient and ion uptake in bacteria . As a group, these systems are related to one another and to other transporters of both prokaryotes and eukaryotes, based on sequence similarity within an ATP-binding subunit and overall structural organization . These transporters probably all use energy derived from ATP to pump substrates across membranes . Although there is considerable information about the sequences and identity of the transporters, there is little information about how they work . That is, where do ligands bind? Where do the subunits or domains interact with one another? How is the energy of nucleotide binding and/or hydrolysis converted to conformational changes? In order to address these questions we have taken a genetic approach that involves studying mutant forms of a transporter . Rather than study mutations that result in complete loss of function, the study of mutations which perturb or alter the normal function of the transporter in a defined manner has provided a limited insight into how the answers to these questions may be obtained. Mol Pharmacol, 1993 Dec, 44(6), 1135 - 41 Nuclear localization of bacterial Streptoalloteichus hindustanus bleomycin resistance protein in mammalian cells; Calmels TP et al.; Prokaryotes produce a variety of toxins that affect genomic function of both eukaryotes and prokaryotes . The 375-base pair bacterial gene Streptoalloteichus hindustanus (Sh) ble encodes a small protein, Streptoalloteichus hindustanus bleomycin resistance protein (BRP), that inhibits in vitro DNA cleavage by the prokaryotic glycopeptide bleomycin, which is a clinically used anticancer drug . NIH/3T3 cells infected with a retroviral vector containing Sh ble (SH-9 cells) were highly resistant to the cytotoxicity of bleomycin-like drugs but not to the cytotoxicity of other, structurally unrelated, DNA-cleaving agents . Expression of BRP did not markedly alter total cellular content or distribution of bleomycin-like compounds . Fluorescently labeled bleomycin was primarily localized in cytoplasmic vesicles in NIH/3T3 and SH-9 cells, whereas BRP, which has no established nuclear localization sequence, was segregated to the nucleus and more specifically to euchromatin . This karyophilic BRP may intercept bleomycin in the nucleus. Dev Biol, 1993 Dec, 160(2), 519 - 34 Embryonic expression of human keratin 18 and K18-beta-galactosidase fusion genes in transgenic mice; Thorey IS et al.; During embryogenesis, EndoB, the mouse form of human keratin 18 (K18), is expressed in a complex spatial and temporal pattern in various embryonic epithelia . We have compared the expression of transgenic human K18 to the endogenous mouse homolog and to the coexpressed, complementary keratin 8 homolog, EndoA, during postimplantation mouse embryogenesis and fetal development in order to determine the developmental expression pattern of the human gene in a mouse environment . The tissue distribution of K18 protein was identical to that of endogenous EndoB in both 7.5- and 13.5-day-old embryos, except for certain heart, eye, and extraembryonic mesodermal tissues in which K18 was not detected . These results indicate that the 10-kb K18 gene specifies appropriate developmental expression in the mouse and support previously reported differences in K18 expression in human and mouse fetal heart . We have also compared the expression patterns of K18 to a series of constructions that utilize the Escherichia coli gene for beta-galactosidase (lacZ) as a reporter gene . Some of these constructions were regulated correctly in embryos during development of the germ layers . However, none was expressed consistently in extraembryonic or in adult tissues . Analysis with methylation-sensitive restriction enzymes revealed that hypermethylation of the CpG-rich prokaryotic reporter gene was not the cause of its silence in adult transgenic liver . However, the repressed state of K18-LacZ transgenes in adult liver was correlated with a different chromatin state that lacked diagnostic DNase hypersensitive sites found in K18 transgenic liver . Expression of the lacZ reporter gene did not accurately reflect the developmental pattern of K18 even in constructions that used all available K18 sequences . We conclude that in these contexts, the lacZ gene was not a developmentally neutral reporter gene. J Infect Dis, 1993 Dec, 168(6), 1422 - 8 Antiinflammatory effects in experimental meningitis of prokaryotic peptides that mimic selectins; Rozdzinski E et al.; The lectin domains of two subunits of pertussis toxin, S2 and S3, share amino acid sequence similarity with the lectin domains of the eukaryotic selectin family . During inflammation, selectins appear on endothelial cells and promote recruitment of leukocytes by reversibly binding carbohydrates . Synthetic peptides representing the carbohydrate recognition domains of S2 and S3 competitively inhibited adherence of neutrophils to endothelial cells in vitro . For some peptides, this antiinflammatory effect occurred without up-regulation of the function of the leukocyte integrin CD11b/CD18 . Intravenous administration of peptides to animals with meningitis disrupted recruitment of leukocytes into the cerebrospinal fluid . These findings indicate that peptides derived from prokaryotic members of the selectin family have therapeutic antiinflammatory potential. Biochemistry, 1993 Nov 30, 32(47), 12555 - 9 Monoselenophosphate: synthesis, characterization, and identity with the prokaryotic biological selenium donor, compound SePX; Glass RS et al.; A labile, selenium donor compound required for synthesis of selenium-dependent enzymes and seleno-tRNAs is formed from ATP and selenide by the SELD enzyme . This compound, tentatively identified as a selenophosphate {Veres, Z., Tsai, L., Scholz, T . D., Politino, M., Balaban, R . S., & Stadtman, T . C . (1992) Proc . Natl . Acad . Sci . U.S.A . 89, 2975-2979}, is indistinguishable from chemically prepared monoselenophosphate by 31P NMR spectroscopy and ion pairing HPLC . Furthermore, addition of chemically prepared monoselenophosphate caused a dose-dependent decrease in the amount of 75Se incorporated into tRNAs from 75SePX generated in situ by SELD enzyme . A procedure is described for the chemical synthesis of monoselenophosphate in which the readily prepared (MeO)3PSe is converted in quantitative yield to (TMSO)3PSe followed by complete cleavage of the latter to monoselenophosphate in oxygen-free aqueous buffer . The chemical properties of chemically synthesized monoselenophosphate are described. Science, 1993 Nov 26, 262(5138), 1407 - 13 A third recognition element in bacterial promoters: DNA binding by the alpha subunit of RNA polymerase; Ross W et al.; A DNA sequence rich in (A+T), located upstream of the -10, -35 region of the Escherichia coli ribosomal RNA promoter rrnB P1 and called the UP element, stimulates transcription by a factor of 30 in vivo, as well as in vitro in the absence of protein factors other than RNA polymerase (RNAP) . When fused to other promoters, such as lacUV5, the UP element also stimulates transcription, indicating that it is a separate promoter module . Mutations in the carboxyl-terminal region of the alpha subunit of RNAP prevent stimulation of these promoters by the UP element although the mutant enzymes are effective in transcribing the "core" promoters (those lacking the UP element) . Protection of UP element DNA by the mutant RNAPs is severely reduced in footprinting experiments, suggesting that the selective decrease in transcription might result from defective interactions between alpha and the UP element . Purified alpha binds specifically to the UP element, confirming that alpha acts directly in promoter recognition . Transcription of three other promoters was also reduced by the COOH-terminal alpha mutations . These results suggest that UP elements comprise a third promoter recognition region (in addition to the -10, -35 recognition hexamers, which interact with the sigma subunit) and may account for the presence of (A+T)-rich DNA upstream of many prokaryotic promoters . Since the same alpha mutations also block activation by some transcription factors, mechanisms of promoter stimulation by upstream DNA elements and positive control by certain transcription factors may be related. Nucleic Acids Res, 1993 Nov 25, 21(23), 5431 - 8 Cloning and expression of the hypoxanthine-guanine phosphoribosyltransferase gene from Trypanosoma brucei; Allen TE et al.; The hypoxanthine-guanine phosphoribosyltransferase (HGPRT) enzyme of Trypanosoma brucei and related parasites provides a rational target for the treatment of African sleeping sickness and several other parasitic diseases . To characterize the T . brucei HGPRT enzyme in detail, the T . brucei hgprt was isolated within a 4.2 kb SalI-KpnI genomic insert and sequenced . Nucleotide sequence analysis revealed an open reading frame of 630 bp that encoded a protein of 210 amino acids with a M(r) = 23.4 kd . After gap alignment, the T . brucei HGPRT exhibited 21-23% amino acid sequence identity, mostly in three clustered regions, with the HGPRTs from human, S . mansoni, and P falciparum, indicating that the trypanosome enzyme was the most divergent of the group . Surprisingly, the T . brucei HGPRT was more homologous to the hypoxanthine phosphoribosyltransferase (HPRT) from the prokaryote V . harveyi than to the eukaryotic HGPRTs . Northern blot analysis revealed two trypanosome transcripts of 1.4 and 1.9 kb, each expressed to equivalent degrees in insect vector and mammalian forms of the parasite . The T . brucei hgprt was inserted into an expression plasmid and transformed into S phi 606 E . coli that are deficient in both HPRT and xanthine-guanine phosphoribosyltransferase activities . Soluble, enzymatically active recombinant T . brucei HGPRT was expressed to high levels and purified to homogeneity by GTP-agarose affinity chromatography . The purified recombinant enzyme recognized hypoxanthine, guanine, and allopurinol, but not xanthine or adenine, as substrates and was inhibited by a variety of nucleotide effectors . The availability of a molecular clone encoding the T . brucei hgprt and large quantities of homogeneous recombinant HGPRT enzyme provides an experimentally manipulable molecular and biochemical system for the rational design of novel therapeutic agents for the treatment of African sleeping sickness and other diseases of parasitic origin. J Biol Chem, 1993 Nov 25, 268(33), 24527 - 30 A small C-terminal region of the Escherichia coli MalT protein contains the DNA-binding domain; Vidal-Ingigliardi D et al.; MalT, the transcriptional activator of the Escherichia coli maltose regulon, is a 901-amino acid-long protein that specifically binds to short, asymmetric nucleotide sequences present in several copies in the promoters of the regulon . We report that the protein structure involved in this specific binding is carried by a small C-terminal part of MalT encompassing the last 95 amino acid residues . This was demonstrated by fusing the last 95 codons of malT to the gene that encodes glutathione S-transferase, purifying the hybrid protein by affinity chromatography, and comparing the DNase I and dimethyl sulfate footprints of the hybrid and of wild-type MalT on different MalT-binding sites . MalT belongs to a large family of prokaryotic transcriptional activators, which share significant homology in their approximately 60-amino acid C-terminal regions . Our result strongly supports the suggestion that the region of homology corresponds to the DNA-binding domain of the proteins in this family. Proc Natl Acad Sci U S A, 1993 Nov 15, 90(22), 10681 - 5 Comparison of somatic mutation in a transgenic versus host locus; Tao KS et al.; Somatic mutations can now be quantified in almost any cell type in mice carrying bacterial genes in a lambda phage shuttle vector . Mutations induced in vivo are detectable ex vivo, after packaging host-cell DNA into phage that are grown on suitable bacteria . However, the transgenic DNA differs from many host loci in several ways: it (i) is prokaryotic DNA, (ii) is present in multiple tandem copies, and (iii) is heavily methylated and probably not expressed . Thus, mutation of a transgene may not be a suitable model of the host loci, which are eukaryotic, unique, and expressed . To test the relevance of the transgene mutation model, the frequencies of the bacterial lacI+ to lacI- mutations induced in half of the small intestine were compared with the frequencies of the host Dlb-1b to Dlb-1a mutations induced in the other half . The loci responded similarly to ethyl nitrosourea (ENU) with respect to the animal's age and sex, sex of the parent transmitting the transgene, and expression time . ENU dose-response curves were similar . Furthermore, no difference was found at the Dlb-1 locus between transgenic and nontransgenic siblings . In contrast, x-rays induced few lacI mutations but many Dlb-1 mutations . Probably few large deletions are detectable at lacI, but many are detectable at Dlb-1 . If so, an important class of mutation is not readily detected in these transgenic mice . With this exception, the transgene and host gene responded similarly in this somewhat limited trial, as is necessary if the transgenic mice are to be a useful model. Proc Natl Acad Sci U S A, 1993 Nov 15, 90(22), 10633 - 7 Expression and biochemical properties of a protein serine/threonine phosphatase encoded by bacteriophage lambda; Barik S; The predicted amino acid sequence encoded by the open reading frame 221 (orf221) of bacteriophage lambda exhibited a high degree of similarity to the catalytic subunits of a variety of protein serine/threonine phosphatases belonging to PP1, PP2A, and PP2B groups . Cloning and expression of the orf221 gene in Escherichia coli provided direct evidence that the gene codes for a protein serine/threonine phosphatase . The single-subunit recombinant enzyme was purified in soluble form and shown to possess a unique repertoire of biochemical properties--e.g., an absolute requirement for Mn2+, resistance to okadaic acid, inhibitors 1 and 2, and ability to dephosphorylate casein, adenovirus E1A proteins, and the alpha subunit of phosphorylase kinase . No phosphotyrosine phosphatase activity was observed . Mutational and biochemical analyses identified the conserved residues 73-77 and Cys138 to be important for activity . The name PP-lambda is proposed for this unusual prokaryotic enzyme. J Immunol, 1993 Nov 15, 151(10), 5742 - 50 Bullous pemphigoid and herpes gestationis autoantibodies recognize a common non-collagenous site on the BP180 ectodomain; Giudice GJ et al.; Bullous pemphigoid (BP) and herpes gestationis (HG) are skin diseases characterized by subepidermal blisters and autoantibodies against two hemidesmosomal Ag, i.e., BP230 and BP180 . Based on sequence analysis the BP180 Ag was predicted to be a transmembrane protein with a long extracellular collagenous domain . In the present investigation fusion proteins encompassing various segments of the BP180 Ag were expressed in a prokaryotic system and assayed by immunoblotting and immunoadsorption against a panel of BP, HG and control sera . One antigenic site, comprising 14 amino acids of the BP180 noncollagenous (NC) 16A domain, was shown to be recognized by 60% of BP sera and by 63% of HG sera tested . 73% (11/15) of BP sera and 100% (8/8) of HG sera reacted with at least one of three BP180 fusion proteins representing various portions of the NC16A domain . Immunoadsorption analysis identified this region of BP180 as an immunodominant site . Using an affinity purified rabbit antiserum raised against a recombinant form of BP180, this BP/HG autoantibody-reactive region was localized to the epidermal basal lamina immediately adjacent to the hemidesmosome . These findings confirmed the predicted type II transmembrane orientation of the BP180 Ag . Thus, the long, C-terminal collagenous domain of this basal keratinocyte protein projects into the basal lamina and may function as a site of interaction with an extracellular matrix component . It is proposed that autoantibodies directed against the well-defined antigenic site on the BP180 ectodomain may play an initiatory role in subepidermal blister formation in BP and HG patients. Biochemistry, 1993 Nov 9, 32(44), 11825 - 37 Crystal structure of ribonuclease Ms (as a ribonuclease T1 homologue) complexed with a guanylyl-3',5'-cytidine analogue; Nonaka T et al.; A ribonuclease T1 homologue, ribonuclease Ms (RNase Ms) from Aspergillus saitoi, has been crystallized as a complex with a substrate analogue GfpC where the 2'-hydroxyl (2'-OH) group of guanosine in guanylyl-3',5'-cytidine (GpC) is replaced by the 2'-fluorine (2'-F) atom to prevent transesterification . The crystal structure of the complex was solved at 1.8-A resolution to a final R-factor of 0.204 . The role of His92 (RNase T1 numbering) as the general acid catalyst was confirmed . Of the two alternative candidates for a general base to abstract a proton from the 2'-OH group, His40 and Glu58 were found close to the 2'-F atom, making the decision between the two groups difficult . We then superposed the active site of the RNase Ms/GfpC complex with that of pancreatic ribonuclease S (RNase S) complexed with a substrate analogue UpcA, a phosphonate analogue of uridylyl-3',5'-adenosine (UpA), and found that His12 and His119 of RNase A almost exactly coincided with Glu58 and His92, respectively, of RNase Ms . Similar superposition with a prokaryotic microbial ribonuclease, RNase St {Nakamura, K . T., Iwahashi, K., Yamamoto, Y., Iitaka, Y., Yoshida, N., & Mitsui, Y . (1982) Nature 299, 564-566}, also indicated Glu58 as a general base . Thus the present comparative geometrical studies consistently favor, albeit indirectly, the traditional as well as the most recent notion {Steyaert, J., Hallenga, K., Wyns, L., & Stanssens, P . (1990) Biochemistry 29, 9064-9072} that Glu58, rather than His40, must be the general base catalyst in the intact enzymes of the RNase T1 family. J Biol Chem, 1993 Nov 5, 268(31), 23376 - 81 Sequence and regulation of the uapA gene encoding a uric acid-xanthine permease in the fungus Aspergillus nidulans; Gorfinkiel L et al.; The nucleotide sequence of the uapA gene, coding for the uric acid-xanthine permease of Aspergillus nidulans, has been determined . The predicted uapA gene product comprises 595 amino acids (M(r) 63,365); it is a highly hydrophobic protein with 12-14 putative transmembrane segments and shows no striking similarity to any other membrane protein of either prokaryotes or eukaryotes, except for a short highly hydrophobic amino acid sequence conserved in a number of different permeases . The presence of an acidic, amphipathic region overlapping with the last hydrophobic segment of UAPA could also be of interest . The results presented suggest that the UAPA permease represents a new type of membrane protein, not described previously . The transcription of uapA is inducible by 2-thiouric acid, and it is highly repressible by ammonium . It is almost absolutely dependent on the presence of functional uaY and areA regulatory gene products . A specific mutation in the GATA binding zinc finger of the AREA protein nearly abolishes uapA transcription . The uap100 cis-acting, up-promoter, constitutive mutation is a duplication that comprises two GATA sites and suppresses weakly the AREA zinc finger mutation but does not alleviate the need for functional UAY and AREA proteins. Toxicon, 1993 Nov, 31(11), 1407 - 14 Identification of protein phosphatase inhibitors of the microcystin class in the marine environment; Chen DZ et al.; Toxins produced by marine phytoplankton represent a severe global health hazard to humans that eat seafood and are also responsible for massive natural fish kills in specialized bloom situations . Tumour-promoting hepatotoxins from the freshwater microcystin/nodularin class were identified in Northeastern Pacific Ocean, Eastern Canadian and European mussels for the first time . These hepatotoxins were detected at biologically active levels up to three-fold higher than accepted quarantine levels for the diarrhetic shellfish toxin okadaic acid (OA), based on their activity (in microcystin-LR equivalent units) in a liquid chromatography (LC)-linked protein phosphatase bioassay . The presence of microcystins/nodularins in oceanic shellfish identifies a potentially novel class of intoxication which is also prevalent in other forms of marine aquatic life, namely sponges and fish . The widespread presence of prokaryotic microcystins and nodularins in the marine environment may be indicative of the importance of signal transduction pathways involving potent inhibition of protein phosphatases in early marine eukaryotes. Protein Eng, 1993 Nov, 6(8), 953 - 64 The expression of bovine microsomal cytochrome b5 in Escherichia coli and a study of the solution structure and stability of variant proteins; Hewson R et al.; The DNA sequence of bovine microsomal cytochrome b5 has been amplified from a liver cDNA library using a polymerase chain reaction . The amplified cDNA when cloned into plasmids that support the high-level production of cytochrome b5 in E.coli leads to protein overexpression and results in cell colonies bearing a strong red colouration . Using cassette mutagenesis, truncated versions of the cytochrome b5 cDNA have been made that encode the first 90 amino acid residues (Ala1-Lys90), the first 104 amino acids (Ala1-Ser104) and the complete protein (Ala1-Asn133) . The location of the overexpressed cytochrome b5 within prokaryotic cells is dependent on the overall length of the protein . Expression of the Ala-Lys90 and Ala1-Ser104 variants leads to a location in the cytoplasmic phase of the bacteria whereas the whole protein, Ala1-Asn133, is found within the bacterial membrane fraction . The last 30 residues of cytochrome b5 therefore contain all of the necessary information to insert the protein into E.coli membranes . The solubility of the Ala1-Ser104 variant permits the solution structure and stability of this protein to be measured using 1- and 2-D 1H-NMR methods and electronic spectroscopy . 1-D NMR studies show that the chemical shifts of the haem and haem ligand resonances of the Ala1-Ser104 variant exhibit only very slight perturbations to their magnetic microenvironments when compared with the tryptic fragment of ferricytochrome b5 . These results indicate an arrangement of residues in the haem pocket that is very similar in both the Ala1-Ser104 variant and the tryptic fragment and by 2-D NMR it is shown that this similarity extends to the conformations of the polypeptide backbone and side chains . Electronic spectroscopy of this variant shows absorbance maxima for the Soret peaks at 423 nm (reduced) and 413 nm (oxidized) . From absorbance spectra the relative thermal stabilities of the Ala1-Ser104 variant and the tryptic fragment were measured . In the oxidized state the Ala1-Ser104 variant denatures in a single cooperative transition with a midpoint temperature (Tm) of 73 degrees C that is significantly higher than that of 'tryptic' ferricytochrome b5 . The reduced form of the protein shows increased transition temperatures (Tm approximately 78 degrees C) reflected in the values of delta Hm, delta Sm and delta(delta G) of 420 kJ/mol, 1096 J/mol/K and 12.38 kJ/mol respectively, estimated for this variant . The increased stability of the Ala1-Ser104 variant and other recombinant forms of cytochrome b5 is correlated with the presence of additional residues at the N- and C-termini.(ABSTRACT TRUNCATED AT 400 WORDS) Mol Biol Evol, 1993 Nov, 10(6), 1239 - 58 Reduced natural selection associated with low recombination in Drosophila melanogaster; Kliman RM et al.; Synonymous codons are not used equally in many organisms, and the extent of codon bias varies among loci . Earlier studies have suggested that more highly expressed loci in Drosophila melanogaster are more biased, consistent with findings from several prokaryotes and unicellular eukaryotes that codon bias is partly due to natural selection for translational efficiency . We link this model of varying selection intensity to the population-genetics prediction that the effectiveness of natural selection is decreased under reduced recombination . In analyses of 385 D . melanogaster loci, we find that codon bias is reduced in regions of low recombination (i.e., near centromeres and telomeres and on the fourth chromosome) . The effect does not appear to be a linear function of recombination rate; rather, it seems limited to regions with the very lowest levels of recombination . The large majority of the genome apparently experiences recombination at a sufficiently high rate for effective natural selection against suboptimal codons . These findings support models of the Hill-Robertson effect and genetic hitchhiking and are largely consistent with multiple reports of low levels of DNA sequence variation in regions of low recombination. J Gen Microbiol, 1993 Nov, 139 ( Pt 11), 2579 - 84 The chromosomal location of genes for elongation factor Tu and ribosomal protein S10 in the cyanobacterium Spirulina platensis provides clues to the ancestral organization of the str and S10 operons in prokaryotes; Sanangelantoni AM et al.; The structural gene (rps10) encoding ribosomal protein S10 of the cyanobacterium Spirulina platensis has been localized both on chromosomal DNA and the previously characterized recombinant plasmid pSp7 harbouring the 3'-terminal portion of the gene for elongation factor G (fus) and the gene for elongation factor Tu (tuf) . Alignment of the predicted S10 sequence of S . platensis with the homologous sequences from cyanelles, bacteria, archaea and eukarya showed that the cyanobacterial S10 shares a high degree of sequence homology (74% amino acid identity) with the cyanellar protein . Unlike the situation in Escherichia coli, the rps10 gene of S . plantensis is unlinked to the S10 operon genes, being adjacent to the str operon genes . Since a similar organization could be observed in cyanelles of Cyanophora paradoxa and in all archaea so far analysed, this probably represents the ancestral state. J Dairy Sci, 1993 Nov, 76(11), 3479 - 89 Supplemental protein influences on carbohydrate degradation and bacterial 16S ribosomal ribonucleic acid; May T et al.; This research examined the mechanism by which soybean protein stimulates growth of mixed ruminal anaerobes and degrades structural polysaccharides in vitro . Soybean meal, isolated soy protein, or branched-chain VFA was added to orchardgrass hay substrate in Experiment 1 . Cell-wall degradation increased 14.5% over that of the control by protein addition . Protein addition resulted in 1.3- to 1.5-fold increases in bacterial growth . Hybridization with a 16S probe specific for Fibrobacter succinogenes indicated that protein addition did not influence the proportion of this species . For in vitro Experiment 2, optimal protein for cell-wall degradation was 2 g/L in cultures containing tall fescue hay . To determine whether protein stimulated microbial colonization of plant cell wall (Experiment 3), orchardgrass hay was placed in 14-L fermentors; treatments were control, NH3 N (2 g of N/L), or isolated soy protein (2 g of N/L) . Addition of protein and NH3 N increased the extent of cell-wall disappearance 9.7% above control . Protein and ammonia improved cell-wall digestion, but protein had the greatest stimulatory effect on prokaryote growth with no preferential effect of F . succinogenes. J Periodontal Res, 1993 Nov, 28(6 Pt 2), 459 - 61 Molecular biology and life science; Matsubara K; Molecular biology has made the transition from phase 1, dealing with prokaryotic genes and DNA, to phase 2, dealing with genes of eukaryotic multicellular organisms . This transition came about because of the DNA technology that has evolved since the early 1970s . Now we are at the beginning of the transition to phase 3, through the emerging human genome project . The background of the development of this project, its current state, and its possible impact on the life sciences is briefly outlined and discussed. J Clin Microbiol, 1993 Nov, 31(11), 3036 - 9 Evaluation of new enzyme-linked immunosorbent assay based on a supernatant containing Staphylococcus aureus alpha-toxin produced by Bacillus subtilis; Egnell P et al.; The gene encoding alpha-toxin from Staphylococcus aureus was cloned into a Bacillus subtilis expression vector (pEF 231/alpha-Tox) . The protease-deficient B . subtilis strain DB 104 transformed with pEF 231/alpha-Tox expressed and secreted 5 mg of alpha-toxin per liter into the growth medium . The alpha-toxin-containing supernatant was diluted 200-fold and used as coating antigen in an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of septicemia and endocarditis caused by S . aureus . Paired sera from patients in acute and convalescent stages of S . aureus and non-S . aureus infections were used to evaluate this ELISA . To evaluate the effectiveness of the crude preparation, the results were compared with those of an ELISA based on a commercially available alpha-toxin . Similar rises in serum titers were obtained with either type of alpha-toxin preparation . This is the first time a crude supernatant without any further purification has been used as an ELISA coating antigen . We therefore conclude that B . subtilis is a suitable host organism for cheap and simple production of prokaryotic recombinant antigens to be used in serodiagnosis. Plant Mol Biol, 1993 Nov, 23(4), 793 - 9 Cytosine deaminase as a negative selective marker for Arabidopsis; Perera RJ et al.; Cytosine deaminase (CD), produced by prokaryotes but not by higher eukaryotes including plants, deaminates cytosine to uracil . The enzyme likewise converts 5-fluorocytosine (5FC), which by itself is not toxic, to 5-fluorouracil (5FU), which is toxic . The Escherichia coli codA-coding sequence encoding CD, together with appropriate regulatory elements, was introduced into Arabidopsis . Neither untransformed controls, nor transgenic plants expressing no CD mRNA, were sensitive to 5FC . Conversely, for most transgenic plants expressing CD mRNA, in the presence of 5FC calli and seedlings failed to proliferate, and seeds failed to germinate . A few transgenic plants with many codA copies expressed less CD mRNA and remained insensitive to 5FC, which likely reflected epigenetic repeat-induced gene silencing . Thus 5FC, presumably through conversion by the enzyme to 5FU, can be used to select against plants that express CD. J Invest Dermatol, 1993 Nov, 101(5), 666 - 72 Treatment of human melanocytes and S91 melanoma cells with the DNA repair enzyme T4 endonuclease V enhances melanogenesis after ultraviolet irradiation; Gilchrest BA et al.; Tanning is a protective response of ultraviolet (UV)-irradiated skin that decreases damage from subsequent sun exposures by increasing the epidermal content of melanin, a brown-black pigment that absorbs light energy throughout the UV and visible portions of the electromagnetic spectrum . The melanin pigment is made by epidermal melanocytes and transferred to surrounding keratinocytes . The action spectrum, time course, and histologic features of tanning are well studied, but the initiating molecular events are unknown . Previous work has shown that T4 endonuclease V, a prokaryotic DNA repair enzyme that catalyzes the first and rate-limiting step in repair of UV-induced pyrimidine dimers, delivered in carrier liposomes (T4N5), enhances repair of UV-induced DNA damage in cultured human cells and protects against photocarcinogenesis in an animal model . We now report that T4N5 treatment enhances UV-induced melanogenesis, as measured by melanin content, tyrosinase activity, 14C-dopa incorporation, and visual assessment in both S91 murine melanoma cells and human melanocytes . T4N5 treatment also increases cell yields following UV irradiation . These data suggest that tanning can be stimulated through enhanced DNA repair. J Bacteriol, 1993 Nov, 175(22), 7282 - 9 The uraA locus and homologous recombination in Mycobacterium bovis BCG; Aldovini A et al.; Molecular genetic manipulation of mycobacteria would benefit from the isolation of mycobacterial genes that could serve both as genetic markers and as sequences used to target homologous integration of recombinant DNA into the genome . We isolated the Mycobacterium bovis BCG gene encoding orotidine-5'-monophosphate decarboxylase (OMP-DCase) by complementing an Escherichia coli mutant defective in this activity . The BCG OMP-DCase gene (uraA) and the flanking DNA were sequenced . The predicted BCG OMP-DCase protein sequence is closely related to the Myxococcus xanthus OMP-DCase and more distantly related to the other known prokaryotic and eukaryotic OMP-DCases . To investigate whether homologous integration can occur in M . bovis BCG, an improved protocol for transformation of BCG was developed and a linear fragment of mycobacterial DNA containing the uraA locus, marked with a kanamycin resistance gene, was introduced into BCG cells by electroporation . The kanamycin-resistant BCG transformants all contained vector DNA integrated into the genome . The marked DNA had integrated into the homologous uraA locus in approximately 20% of the transformants . These results have implications for understanding the role of mycobacterial genes in disease pathogenesis and for the genetic engineering of improved mycobacterial vaccines. J Bacteriol, 1993 Nov, 175(21), 6908 - 15 SKN7, a yeast multicopy suppressor of a mutation affecting cell wall beta-glucan assembly, encodes a product with domains homologous to prokaryotic two-component regulators and to heat shock transcription factors; Brown JL et al.; A search for genes which, at elevated copy number, could suppress the growth defect in a strain disrupted at the KRE9 locus has identified the SKN7 gene . SKN7 was mapped to the right arm of chromosome VIII and is predicted to encode a 70-kDa protein, Skn7p, with a region of homology to the DNA binding domain of the Saccharomyces cerevisiae heat shock transcription factor, Hsf1p . Skn7p also has a domain which shows similarity to the prokaryotic receiver modules found on an extensive family of two-component response regulators, including the products of the rcsC and barA genes . SKN7 did not suppress other mutations in the (1-->6)-beta-glucan biosynthetic pathway, suggesting that SKN7 does not act as a general bypass suppressor of this glucan. Exp Parasitol, 1993 Nov, 77(3), 282 - 94 Schistosoma mansoni: immunoreactivity of human sera with the surface antigen Sm23; Koster B et al.; Sm23, a surface antigen of Schistosoma mansoni, elicits an immune response in patients infected with the parasite, although to a lesser degree than Sj23, the homologue of Sm23 in Schistosoma japonicum . To characterize the host immune response to Sm23, we have expressed full-length Sm23 using the baculovirus expression system and have also expressed the N-terminal 133 amino acids and the 85 C-terminal amino acids by means of the prokaryotic vector pGEX . A total of 70 sera from patients from Sudan and Egypt were examined for alpha-Sm23 antibodies in an ELISA and in Western blot analysis using both the C-terminal polypeptide and the full-length protein . A subset of these sera was also tested for reactivity with the N-terminal polypeptide . The alpha-Sm23 antibody titers in infected patients varied widely and were not correlated with egg counts or age of the individuals . Most of the seroreactivity was directed against the C-terminal polypeptide. Virology, 1993 Nov, 197(1), 468 - 70 Detection of the L protein of tomato spotted wilt virus; van Poelwijk F et al.; The 5'-terminal and 3'-terminal parts of the single open reading frame (ORF) in the L RNA of tomato spotted wilt virus (TSWV) were expressed using a prokaryotic expression system . Using antibodies raised against the translational products obtained a 330-kDa protein could be specifically detected in preparations of purified virions and in nucleocapsid preparations from TSWV-infected leaf tissue . The results obtained indicate that the L protein of TSWV, though much larger than that of the animal-infecting bunyaviruses, is present in virus particles in an unprocessed, intact form. Allergy, 1993 Nov, 48(8), 615 - 23 Nucleotide sequence of cD