Prokaryotes

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 cDNA encoding the group II allergen of cocksfoot/orchard grass (Dactylis glomerata), Dac g II; Roberts AM et al.; Cocksfoot/orchard grass (Dactylis glomerata) anther cDNA clones encoding the group II allergen Dac g II were previously isolated on the basis of immunoreactivity of human, rabbit, and murine antibodies with a 24-kDa protein expressed as a fusion protein with beta-galactosidase . Nucleotide sequencing reveals an open reading frame predicting expression of a 98-amino-acid (11-kDa) polypeptide exhibiting > 90% homology with the group II allergen of Lolium perenne, Lol p II . In vitro translation of different sized clone fragments generated by polymerase chain amplification confirms eukaryotic expression of a 10-12-kDa polypeptide by SDS-PAGE and the position of a translational stop apparently unrecognized during expression of lambda gt11 in E . coli . The unusual characteristics of the prokaryote-expressed fusion proteins may be exerting conformational alterations in Dac g II, as reflected by previous demonstrations of differences in human IgE immunoreactivity . Northern blot analysis using PCR-generated partial and full-length probes suggests that group II allergens may be encoded by a different family or families of temporally expressed genes from those encoding group I major allergens, although a group I gene may have been the progenitor.

Mol Microbiol, 1993 Nov, 10(3), 465 - 71
DNA rearrangements and phenotypic switching in prokaryotes; Dybvig K; Microorganisms have numerous strategies for coping with environmental changes . In many systems, a single cell has the capacity to generate a seemingly infinite array of phenotypic variants in just a few generations of growth . The resulting heterogeneous population is well equipped for sudden environmental change; even if only a few cells in the population possess a phenotype needed for survival, these cells have the capacity to regenerate a similarly diverse population . Phenotypic switching in these systems usually results from high-frequency DNA rearrangements which are the subject of this review.

Mol Cell Biochem, 1993 Nov, 127-128, 71 - 80
Expression of cGMP-dependent protein kinase in Escherichia coli; Feil R et al.; Cyclic GMP-dependent protein kinase (cGMP kinase) is involved in the relaxation of smooth muscle . The enzyme has been cloned and expressed in eukaryotic cell lines but so far not in prokaryotic cells . Three vectors were constructed for the expression of I alpha cGMP kinase in Escherichia coli . Transformation with the pET3a/cgk vector which uses the T7 RNA polymerase/promotor system resulted in efficient accumulation of cGMP kinase . Most of the protein was in an insoluble and catalytic inactive form . Various solubilization and refolding conditions did not yield an active enzyme . A small fraction of the cGMP kinase was present in the soluble cell extract . This fraction bound cGMP with high affinity but had no cGMP stimulated kinase activity . To prevent aggregation two additional vectors were constructed . (I) A bacterial leader sequence, which directs the export of proteins into the periplasmic space, was fused to the amino-terminus of the cGMP kinase . (II) A gram/gram+ shuttle vector for expression under the control of the tac promotor was used . Both constructs directed the synthesis of an insoluble and inactive cGMP kinase . These results suggest that large amounts of cGMP kinase can be expressed in E . coli, but mainly in an insoluble and inactive form . In contrast to eukaryotic cells, bacteria may lack systems for correct protein folding and/or posttranslational modification that are crucial for the productive folding and/or activation of cGMP kinase.

Trends Biochem Sci, 1993 Nov, 18(11), 442 - 6
Intramolecular chaperones and protein folding; Shinde U et al.; Many proteins from both prokaryotic and eukaryotic sources are produced with amino-terminal propeptides . These propeptides, which are usually located between the signal peptide and the mature protein, are essential for the proper function of that protein . Recent research has indicated that these polypeptides are indispensible for proper folding of the proteins they are attached to . As propeptides perform a function similar to that of a large family of heat shock proteins, they had been broadly classified as molecular chaperones . However, significant differences exist between these two classes of proteins and to distinguish them from one another, propeptides have been termed intramolecular chaperones . Recent results have suggested that such intramolecular chaperones may be found in a large number of proteins.

Int J Radiat Biol, 1993 Nov, 64(5), 613 - 9
DNA excision repair as a component of adaptation to low doses of ionizing radiation in Escherichia coli; Huang H et al.; In this study we examined whether or not DNA excision repair is a component of adaptation induced by very low-dose ionizing radiation in Escherichia coli, a well-characterized prokaryote, and investigated the relationship between enhanced excision repair and the SOS response . Competent E . coli cells were irradiated using low doses (0.1-10 Gy) of 137Cs gamma-rays, allowed to recover for 2 h and were then transformed using pUC18 DNA containing approximately 22 oxidized thymine residues (thymine glycols) per molecule . Successful transformants were identified by recovery of plasmid-borne ampicillin resistance and the resulting data (colony counts) were used to calculate ratios of plasmid recovery in irradiated to control cells . Results showed that cells irradiated with very low doses (0.1-0.5 Gy) were up to 30-40% more efficient at utilizing thymine glycol-containing pUC18 DNA . The enhanced excision repair by very low doses (< 0.5 Gy) of gamma-rays was shown to be independent of the recA-controlled SOS response in experiments using recA cells or cells carrying recA-lacZ gene fusions . The stimulating effect in AB1157 prototype cells were subsequently confirmed using a DNA precipitation assay in which DNA incision events were accumulated by inhibiting DNA ligation with ethidium bromide . Our data suggest that there seems to be narrow 'windows' of dose-effect for the induction of SOS-independent DNA excision repair . Being similar to mammalian cell studies, the dose range for this effect was about 200-fold less than D37 for radiation survival.

Trends Biochem Sci, 1993 Nov, 18(11), 419 - 23
What makes an mRNA anti-sense-itive?
Nellen W, Lichtenstein C.
Antisense RNA has been used for some time as a versatile tool for silencing gene expression . There is ample evidence for gene regulation by endogenous antisense transcripts in prokaryotes and increasing insight into the molecular mechanisms underlying such regulation . The introduction of antisense gene constructs into eukaryotes has now become routine but the mechanisms by which gene expression is inhibited are barely understood . In recent years, several examples of endogenous eukaryotic antisense transcripts have been discovered, some of which probably serve regulatory functions . Here we will discuss a model to explain mechanisms of antisense-mediated gene silencing.

Mol Biol Evol, 1993 Nov, 10(6), 1343 - 59
Contrasting evolutionary rates in the duplicate chaperonin genes of Mycobacterium tuberculosis and M . leprae; Hughes AL; A phylogenetic analysis of chaperonin (heat shock protein 60) sequences from prokaryotes and eukaryotes indicated that a single gene duplication event in the common ancestor of Mycobacterium tuberculosis, M . leprae, and Streptomyces albus gave rise to the duplicate chaperonin genes found in these species (designated HSP65 and GroEL in the mycobacterial species) . Comparison of rates of synonymous and nonsynonymous nucleotide substitution in different gene regions suggested that the 5' end of the HSP65 gene was homogenized by an ancient recombination event between M . tuberculosis and M . leprae . In S . albus, the two duplicated chaperonin genes have evolved at essentially the same rate . In both M . tuberculosis and M . leprae, however, the GroEL gene has evolved considerably more rapidly at nonsynonymous nucleotide sites than has the HSP65 gene . Because this difference is not seen at synonymous sites, it must be due to a difference in selective constraint on the proteins encoded by the two genes, rather than to a difference in mutation rate . The difference between GroEL and HSP65 is striking in regions containing epitopes recognized by T cells of the vertebrate host; in certain cross-reactive epitopes conserved across all organisms, nonsynonymous sites in GroEL have evolved twice as fast as those in HSP65 . It is suggested that these differences are correlated with differences in the way in which the duplicate chaperonins of M . tuberculosis and M . leprae interact with the host immune system.

Science, 1993 Oct 22, 262(5133), 539 - 44
Arabidopsis ethylene-response gene ETR1: similarity of product to two-component regulators; Chang C et al.; Ethylene behaves as a hormone in plants, regulating such aspects of growth and development as fruit ripening, flower senescence, and abscission . Ethylene insensitivity is conferred by dominant mutations in the ETR1 gene early in the ethylene signal transduction pathway of Arabidopsis thaliana . The ETR1 gene was cloned by the method of chromosome walking . Each of the four known etr1 mutant alleles contains a missense mutation near the amino terminus of the predicted protein . Although the sequence of the amino-terminal half of the deduced ETR1 protein appears to be novel, the carboxyl-terminal half is similar in sequence to both components of the prokaryotic family of signal transducers known as the two-component systems . Thus, an early step in ethylene signal transduction in plants may involve transfer of phosphate as in prokaryotic two-component systems . The dominant etr1-1 mutant gene conferred ethylene insensitivity to wild-type Arabidopsis plants when introduced by transformation.

FEBS Lett, 1993 Oct 18, 332(3), 203 - 7
Redox control of transcription: sensors, response regulators, activators and repressors; Allen JF; In a growing number of cases, transcription of specific genes is known to be governed by oxidation or reduction of electron carriers with which the gene products interact . The biological function of such control is to activate synthesis of appropriate redox proteins, and to repress synthesis of inappropriate ones, in response to altered availability of specific electron sources and sinks . In prokaryotic systems this control appears to operate by two general classes of mechanism: by two-component regulation involving protein phosphorylation on histidine and aspartate; and by direct oxidation-reduction of gene repressors or activators . For the first class, termed 'two-component redox regulation', the term 'redox sensor' is proposed for any electron carrier that becomes phosphorylated upon oxidation or reduction and thereby controls phosphorylation of specific response regulators, while the term 'redox response regulator' is proposed for the corresponding sequence-specific DNA-binding protein that controls transcription as a result of its phosphorylation by one or more redox sensors . For the second class of redox regulatory mechanism, the terms 'redox activator protein' and 'redox repressor protein' are proposed for single proteins containing both electron transfer and sequence-specific DNA-binding domains . The structure, function and biological distribution of these components are discussed.

J Biol Chem, 1993 Oct 15, 268(29), 22175 - 80
Characterization of a chloroplast homologue of the 54-kDa subunit of the signal recognition particle; Franklin AE et al.; Proteins and RNAs homologous to components of the eukaryotic signal recognition particle (SRP) have been identified in a number of prokaryotic organisms . Here we report the isolation of an Arabidopsis thaliana cDNA, FFC (fifty-four chloroplast homologue), that encodes a chloroplast protein (54CP) homologous to the 54-kDa subunit of the signal recognition particle . 54CP shares 27% identity with mammalian SRP54 and 44% identity with the Escherichia coli ffh gene product suggesting a prokaryotic origin for this gene . FFC is a nuclear gene encoding a 62-kDa cytoplasmically synthesized precursor that is capable of being imported into isolated chloroplasts and processed to a 53-kDa stromal polypeptide . Antibodies generated against a portion of 54CP recognize an endogenous 53-kDa chloroplast protein that sediments at 4 S and 70 S, the latter form may be associated with ribosomes . The FFC transcript is most abundant in green shoot tissue whereas etiolated buds and roots have transcript levels about 30 and 10%, respectively, relative to light grown green shoots . By analogy with the eukaryotic signal recognition particle which targets secretory proteins to the endoplasmic reticulum, it is speculated that 54CP may target chloroplast proteins to either the thylakoid or envelope membranes.

J Immunol, 1993 Oct 15, 151(8), 3971 - 80
Alkali hydrolysis of recombinant proteins allows for the rapid identification of class I MHC-restricted CTL epitopes; Gavin MA et al.; The characterization of the epitopes recognized by CTL provides insights into the nature of protective immune responses and facilitates the development of methods to enhance immunity to human pathogens . However, no easily applicable approach for CTL epitope identification has been developed . We present a rapid and efficient method for locating CTL epitopes within a protein . The gene encoding the protein of interest is inserted into an inducible prokaryotic expression vector . Random peptides are then generated by alkali digestion of intact or lysed Escherichia coli expressing the protein and assayed for the presence of the epitope by coating target cells for a standard CTL targeting assay . A large panel of clones containing serial 3'-deletions of the gene is then generated by exonuclease III digestion, and the expressed truncated proteins are similarly analyzed for the presence of the antigenic peptide . The epitope is located by determining the deletion points of clones expressing sequential truncations and differing in Ag expression . This technique was used to identify the H-2Ld-restricted nonamer in E . coli beta-galactosidase, with residues 876-884 representing the naturally processed epitope . To test the applicability of this method to other proteins, two genes from human CMV, an often fatal pathogen in immunocompromised individuals, were screened for HLA class I-restricted epitopes . An HLA-B18-restricted epitope from the CMV major immediate-early protein was found to lie between residues 378 and 389, and an HLA-B35-restricted epitope from the CMV pp65 matrix protein was characterized as residues 123 to 131 . The results demonstrate that this technique can be used to rapidly identify CTL epitopes within a chosen protein and should be useful for assaying viral isolates or neoplasms for loss of epitopes after mutation and selection by host immune responses.

Biochemistry, 1993 Oct 12, 32(40), 10905 - 11
Identity of the axial ligand of the high-spin heme in cytochrome oxidase: spectroscopic characterization of mutants in the bo-type oxidase of Escherichia coli and the aa3-type oxidase of Rhodobacter sphaeroides; Calhoun MW et al.; Prokaryotic and eukaryotic cytochrome c oxidases and several bacterial ubiquinol oxidases compose a superfamily of heme-copper oxidases . These enzymes are terminal components of aerobic respiratory chains, the principal energy-generating systems of aerobic organisms . Two such heme-copper oxidases are the aa3-type cytochrome c oxidase of Rhodobacter sphaeroides and the bo-type ubiquinol oxidase of Escherichia coli . These enzymes catalyze the reduction of oxygen to water at a heme-copper binuclear center . Energy conservation is accomplished by coupling electron transfer through the metals of the oxidases to proton translocation across the cellular membrane . The Rb . sphaeroides and E . coli enzymes have previously been utilized in site-directed mutagenesis studies which identified two histidines which bind the low-spin heme (heme a), as well as additional histidine residues which are probable ligands for copper (CuB) . However, the histidine that binds the heme of the binuclear center (heme a3) could not be unequivocally identified between two residues (His284 and His419) . Additional characterization by Fourier transform infrared spectroscopy of the CO-bound forms of the E . coli enzyme in which His284 is replaced by glycine or leucine demonstrates that these mutations cause only subtle changes to CO bound to the heme of the binuclear center . Resonance Raman spectroscopy of the Rb . sphaeroides enzyme in which His284 is replaced by alanine shows that the iron-histidine stretching mode of heme a3 is maintained, in contrast with the loss of this mode in mutants at His419 . These results demonstrate that His284 is not the heme a3 ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

J Natl Cancer Inst, 1993 Oct 6, 85(19), 1558 - 70
Biological and clinical implications of heat shock protein 27,000 (Hsp27): a review; Ciocca DR et al.; Heat shock and other environmental and pathophysiologic stresses stimulate synthesis of heat shock proteins (Hsps) . These proteins enable the cell to survive and recover from stressful conditions by as yet uncompletely understood mechanisms . Hsp27 is an important small Hsp (molecular weight, 27,000) found in human cells--both cancer cells and normal cells . This protein, besides its putative role in thermotolerance, is of special clinical interest because of recent data suggesting it may also play a role in drug resistance . In adults, Hsp27 is found particularly in several cell types such as breast, uterus, cervix, placenta, skin, and platelets . Although low-molecular-weight (small) Hsps have been found to be involved in embryogenesis of Xenopus and Drosophila, they have not been detected in human fetal organs . Regulation of expression of the Hsp gene (also known as HSPB1) has been considered a paradigm of gene regulation and is actively being studied in both prokaryotes and eukaryotes . In prokaryotes, the major Hsp genes are transcriptionally regulated by positively and negatively acting transcription factors . In eukaryotes, the genes encoding Hsps contain a regulatory DNA motif (inverted repeats of the pentameric sequence nGAAn) known as the heat shock element . Hsp27 may function as a molecular chaperone and in signal transduction pathways of different cell regulators, and Hsp27 and other Hsps may be active in development of resistance to stressful conditions and agents including cytotoxic drugs . Study findings indicate that some but not all estrogen-positive breast cancers express Hsp27, and overexpression of Hsp27 has been associated with both good and poor prognosis . In endometrial carcinomas, the presence of Hsp27 is correlated with the degree of tumor differentiation as well as with the presence of estrogen and progesterone receptors . Studies suggest, however, that detection of Hsp27 should not be considered to be a method for identifying hormone-responsive tumors or detecting estrogen receptors . Hsp27 seems to be a biochemical marker of estrogenic endometrial response . In patients with cervical cancer, Hsp27 is predominantly expressed in well-differentiated and moderately differentiated squamous cell carcinomas . In addition, expression of Hsp27 seems to be a negative prognostic factor for gastric cancer . Different isoforms of Hsp27 have been found in lymphoid tissue of patients with acute lymphoblastic leukemia, and the protein has also been associated with viral infections . These aspects are summarized and discussed in the present review.

Genes Dev, 1993 Oct, 7(10), 2016 - 32
The immunoglobulin mu enhancer core establishes local factor access in nuclear chromatin independent of transcriptional stimulation; Jenuwein T et al.; Factor access in chromatin has been proposed to be facilitated by transcriptional enhancers . With the aim of uncoupling factor access from transcriptional stimulation by protein-protein contacts, we analyzed the potential of enhancer fragments to confer accessibility upon a linked promoter for prokaryotic T7 RNA polymerase . Access to the T7 promoter in pre-B cells from transgenic mice was examined by transcribing chromatin of isolated nuclei with T7 RNA polymerase . A 95-bp immunoglobulin mu enhancer core element was necessary and sufficient to confer accessibility upon the T7 promoter independent of its chromosomal position . This enhancer-dependent factor access could be uncoupled from an active transcriptional state of the transgene and was not accompanied by the formation of pronounced DNase I hypersensitive sites . Additional mu enhancer sequences comprising previously identified matrix attachment regions and a cryptic promoter were required to induce DNase I hypersensitivity . Together, these data provide evidence that the 95-bp mu enhancer core can establish localized factor access in nuclear chromatin independent of detectable transcription by endogenous polymerases and suggest that multiple steps are involved in the alteration of chromatin structure.

EMBO J, 1993 Oct, 12(10), 3957 - 65
Transcription of ftsZ oscillates during the cell cycle of Escherichia coli; Garrido T et al.; The FtsZ protein is a key element controlling cell division in Escherichia coli . A powerful transcription titration assay was used to quantify the ftsZ mRNA present in synchronously dividing cells . The ftsZ mRNA levels oscillate during the cell cycle reaching a maximum at about the time DNA replication initiates . This cell cycle dependency is specifically due to the two proximal ftsZ promoters . A strain was constructed in which expression of ftsZ could be modulated by an exogenous inducer . In this strain cell size and cell division frequency were sensitive to the cellular FtsZ contents, demonstrating the rate-limiting role of this protein in cell division . Transcriptional activity of the ftsZ promoters was found to be independent of DnaA, indicating that DNA replication and cell division may be independently controlled at the time when new rounds of DNA replication are initiated . This suggests a parallelism between the prokaryotic cell cycle signals and the START point of eukaryotic cell cycles.

DNA Cell Biol, 1993 Oct, 12(8), 763 - 70
DNA polymerase-catalyzed addition of nontemplated extra nucleotides to the 3' end of a DNA fragment; Hu G; Some prokaryotic and eukaryotic DNA polymerases are capable of adding an additional nontemplated nucleotide residue at the 3' end of a DNA fragment (Clark et al., 1987; Clark, 1988) . The extra nucleotide at the 3' end of the PCR product has been shown to be a critical factor determining the efficiency of cloning PCR products into plasmids and can affect mutation analyses with a PCR-denaturing gradient gel electrophoresis (DGGE) approach (Pfeiffer and Hu, 1993) . In the present work, the ability of various DNA polymerases to add an extra nontemplated nucleotide at the 3' end of DNA was studied . The results show that out of the eight studied enzymes, five can add, with varying efficiencies, an extra nucleotide residue at the 3' end of DNA . Which extra nucleotide is added depends on the terminal residue and the DNA polymerase . Among the enzymes, thermostable Pfu DNA polymerase is found to be the best choice for PCR due to its relatively high fidelity (Scott et al., 1991; Coller, unpublished), and ability to produce blunt-ended DNA fragments . The relationship between the DNA polymerases' ability to add an extra nucleotide and their 3'-->5' exonuclease activity is also discussed.

Virology, 1993 Oct, 196(2), 557 - 63
Moloney murine leukemia virus protease: bacterial expression and characterization of the purified enzyme; Menendez-Arias L et al.; The Moloney murine leukemia virus (Mo-MuLV) protease has been cloned into the prokaryotic expression vector pGEX-2T, expressed in fusion with the glutathione S-transferase from Schistosoma japonicum, and purified to apparent homogeneity after thrombin cleavage of the chimeric protein . The purified protease showed maximum activity at pH 6.0 and was inhibited by several aspartyl protease inhibitors, found to be active toward the human immunodeficiency virus-1 (HIV-1) protease . Peptides representing maturation cleavage sites in Gag and Gag-Pol polyproteins were accurately cleaved by the recombinant protease, and kinetic parameters have been determined . In addition, oligopeptides mimicking the cleavage site found in the transmembrane protein and leading to the formation of p15E and p2E were also hydrolyzed at the expected position . The Mo-MuLV protease appears to be more closely related to the HIV-1 protease than to the mouse mammary tumor virus enzyme, based on its substrate specificity and sensitivity to aspartyl protease inhibitors.

J Antimicrob Chemother, 1993 Oct, 32(4), 539 - 50
A study on the mechanism of action of sceptrin, an antimicrobial agent isolated from the South Pacific sponge Agelas mauritiana; Bernan VS et al.; The mechanism of action of sceptrin, an antimicrobial agent isolated from the sponge Agelas mauritiana, was investigated . Sceptrin has been reported to exhibit antibacterial and antifungal activities . In our studies, sceptrin demonstrated a bacteriostatic rather than bactericidal effect on exponentially growing Escherichia coli cells at the MIC . Under these conditions, the culture produced chains of cells, and incorporation of radio-labelled precursors into DNA, protein, and cell wall was unaffected, whereas incorporation of 3H-uridine into RNA was slightly inhibited . At concentrations higher than the MIC, sceptrin was bactericidal, inhibited the incorporation of all radiolabelled precursors, and induced the formation of unusual spheroplasts . Peptidoglycan turnover in E . coli appeared to be stimulated by sceptrin as demonstrated by a release of diaminopimelic acid-containing high molecular weight material . Subsequent studies of the release of potassium ions from E . coli and the lysis of red blood cells suggested that sceptrin disrupts the cell membranes of both prokaryotic and eukaryotic cells . It is proposed that spheroplasts formation may reflect a cell wall effect that occurs subsequent to membrane damage.

Protein Expr Purif, 1993 Oct, 4(5), 438 - 44
Cofactor identification of threonine-serine dehydratase from sheep liver; Tong H et al.; L-Threonine-serine dehydratase catalyzes the conversion of L-threonine and L-serine to alpha-ketobutyric acid and pyruvate, respectively . The enzyme has been purified to homogeneity from extracts of sheep liver . In the past, various cofactors have been suggested for threonine dehydratase from both prokaryotic and eukaryotic tissue . While some direct evidence for the presence of pyriodoxal 5'-phosphate in impure preparations is present in the literature no direct evidence for the cofactor in homogeneous dehydrogenase from mammalian tissue has been reported . The threonine dehydratase of sheep liver has been obtained in a homogeneous form and a spectral study provides clear evidence for the presence of pyridoxal 5'-phosphate . Both the physical properties of homogeneous threonine dehydratase and a study of spectral properties of its cofactor are reported in this communication.

Lipids, 1993 Oct, 28(10), 877 - 82
pH-dissociation characteristics of cardiolipin and its 2'-deoxy analogue; Kates M et al.; Cardiolipin (CL) is found in inner mitochondrial membranes and the plasma membrane of aerobic prokaryotes . CL is tightly bound to those transmembrane enzymes associated with oxidative phosphorylation . CL has earlier been reported to have a single pK at low pH . We have titrated CL in aqueous suspension (bilayers) and in solution in methanol/water (1:1, vol/vol) and found it to display two different pK values, pK1 at 2.8 and pK2 initially at 7.5 but shifting upwards to 9.5 as the titration proceeds . The unusually high pK2 might be explained by the formation of a unique hydrogen bond in which the free hydroxyl on the central glycerol forms a cyclic intramolecular hydrogen-bonded structure with one protonated phosphate (P-OH group) . We have therefore chemically synthesized the 2'-deoxycardiolipin analogue, which lacks the central free hydroxyl group, and measured its pH-dissociation behavior by potentiometric titration, under the same conditions as those for CL . The absence of the hydroxyl group changes the titration dramatically so that the deoxy analogue displays two closely spaced low pK values (pK1 = 1.8; pK2 = 4.0) . The anomalous titration behavior of the second dissociation constant of CL may be attributed to the participation of the central glycerol OH group in stabilizing the formation of a cyclic hydrogen-bonded monoprotonated form of CL, which may function as a reservoir of protons at relatively high pH . This function may have an important bearing on proton pumping in biological membranes.

Mol Biol Rep, 1993 Oct, 18(3), 223 - 30
Affinity purification of histidine-tagged proteins; Schmitt J et al.; Expression of recombinant proteins is a standard technique in molecular biology and a wide variety of prokaryotic as well as eukaryotic expression systems are currently in use . A limiting step is often the purification of the expressed recombinant protein, particularly if mammalian expression systems that yield low expression levels are employed . Here, we discuss the advantages and restrictions of tagging recombinant proteins with histidines and purifying them by Ni(2+)-NTA chromatography.

Infect Immun, 1993 Oct, 61(10), 4158 - 66
Molecular and immunological characterization of a novel polymorphic lipoprotein of Borrelia burgdorferi; Wallich R et al.; We describe the cloning, expression, and molecular characterization of a novel polymorphic Borrelia burgdorferi lipoprotein recognized by monoclonal antibody LA7 . Sequence analysis revealed an open reading frame encoding a 21,866-Da polypeptide (IpLA7) . Comparison with other known proteins indicated sequence similarity between IpLA7 signal peptides and those of other prokaryotic lipoproteins, including the immunodominant B . burgdorferi outer surface proteins OspA, OspB, pC, and OspD . Both natural IpLA-7 and recombinant IpLA-7 could be biosynthetically labeled with {3H}palmitate . Upon solubilization of intact B . burgdorferi with the nonionic detergent Triton X-114, IpLA7 was extracted together with other lipoproteins into the detergent phase . Indirect immunolabeling studies indicated that the epitope recognized by monoclonal antibody LA7 is mainly located in the periplasmic space . Two-dimensional gel electrophoresis and immunoblotting confirmed the calculated acidic pI of 5.7 for IpLA-7 . The LA7 gene was shown to be species specific and to be located on the linear chromosome of B . burgdorferi . The analysis of 40 individual spirochetal isolates on the basis of restriction fragment length polymorphisms revealed considerable genotypic heterogeneity of LA7 corresponding to that previously found for ospA . Native IpLA-7 and recombinant IpLA-7 were recognized by immune sera from infected mice as well as some human sera derived from infected but healthy donors and may thus prove useful as an additional marker for the serodiagnosis of Lyme disease.

Rev Latinoam Microbiol, 1993 Oct-Dec, 35(4), 415 - 22
{Use of gene fusion in the study of the interaction of ribosomal protein L45 with the Saccharomyces cerevisiae ribosome}; Santana-Roman H et al.; Cytoplasmic ribosomes from all cells have a set of highly acidic proteins (proteins A; pI less than 4.0) . Proteins A are indispensable for ribosomal function since they mediate the interaction between GTP dependent translational factors and the ribosome . From gene cloning and DNA sequence analysis, during the past few years the primary structure of several prokaryotic and eukaryotic proteins A has been deduced . When their primary structure is compared, it is evident that these proteins have three well defined domains: firstly the aminus terminal domain, comprising the first 40 to 50 amino acids; secondly, the central domain, rich in non-polar amino acids and a high percent of alpha helix; and thirdly, the alpha carboxyl terminal domain, relatively acidic and highly conserved throughout evolution . In Saccharomyces cerevisiae the four genes coding for proteins A L45, L44, L44' and A1 have been cloned and sequenced . This enabled us to study the physiological role of each of the three domains in proteins A . Plasmid YEp45-1 encompassing a 596 base pairs stretch of the L45 genomic DNA fused to the LacZ gene was constructed and used to transform yeast cells . The chimeric gene expressed an hybrid protein in which the covalently linked beta-galactosidase enzyme follows after the first 75 amino acids from the alpha amino terminal domain of protein A L45 . After cell fractionation of yeast transformants, beta-galactosidase activity was found specifically bound to the 80S ribosome and polysome particles . From these results we conclude that the amino terminal domain of protein L45 interacts to the ribosome . We postulate, by inference, that the carboxyl terminal domain of proteins A interacts with the GTP dependent cytoplasmic translational factors.

Mol Microbiol, 1993 Oct, 10(1), 99 - 111
Characterization of comE, a late competence operon of Bacillus subtilis required for the binding and uptake of transforming DNA; Hahn J et al.; The binding and transport of DNA by competent Bacillus subtilis requires the assembly of a specialized apparatus . We present here the characterization of comE, an operon under competence control that is required for both DNA binding to the competent cell surface, and for uptake . comE contains three open reading frames (ORF1-3) read in the forward direction, preceded by a long untranslated leader sequence and an apparent E sigma A promoter . A minor promoter also is responsible for transcription of ORF2 and -3 . A transcript containing a single ORF is produced in the reverse direction . The reverse ORF overlaps ORF1 and the untranslated comE leader . The comE transcript is present at a very low level during growth and at an elevated level in stationary-phase cells . Conversely, the reverse transcript is present during exponential growth and disappears during stationary phase . The reverse ORF resembles prokaryotic and eukaryotic pyrroline-5'-carboxylate reductases, while ORF2 is similar to several dCMP deaminases . ORF1 and ORF3 are predicted to be integral membrane proteins . The latter is specifically required for DNA uptake but not for binding.

Mol Microbiol, 1993 Oct, 10(1), 57 - 62
Retrotransfer of IncP plasmid R751 from Escherichia coli maxicells: evidence for the genetic sufficiency of self-transferable plasmids for bacterial conjugation; Heinemann JA et al.; Gene transfer between organisms is a prime contributor to evolution . Bacterial conjugation is probably the most important mechanism by which genes are spread among prokaryotes and perhaps also contributes to eukaryotic evolution . Conjugation is mediated by plasmids . The mechanism of conjugation remains ill-understood despite progress in the identification, mapping and sequencing of genes required for plasmid transmission . All conjugation-specific genes (those required only for DNA transfer and establishment) identified to date map to plasmids . We found that IncP plasmids could enter and subsequently convert maxicells, which are trapped in a metabolic state that prevents de novo expression of chromosomal genes, into conjugative donors . This suggests that IncP plasmids encode not only necessary functions but indeed all functions specific to DNA transmission . Thus, like viruses, plasmids can convert non-viable cells into gene vectors.

Mol Microbiol, 1993 Oct, 10(2), 421 - 30
Cytochrome bd biosynthesis in Escherichia coli: the sequences of the cydC and cydD genes suggest that they encode the components of an ABC membrane transporter; Poole RK et al.; At least four genes are known to affect formation of the cytochrome bd-type terminal oxidase of Escherichia coli . In addition to the genes (cydA and cydB) encoding the two constituent subunits of this complex, a further two genes (cydC and cydD) map near 19 min on the E . coli chromosome . We report here the cloning of both genes on a 5.3 kb ClaI-HindIII restriction fragment, which, when used to transform either a cydC or cydD mutant, restored the ability of these mutants to grow on a selective medium containing azide and zinc ions and also restored the spectral signals associated with the cytochrome components of the oxidase complex . A subcloned 1.8 kb DdeI fragment similarly restored growth and cytochrome content of a cydD mutant, but not a cydC mutant . The complete nucleotide sequence of the ClaI-HindIII fragment reveals three open reading frames, one being trxB (19.3 min on the E . coli chromosome map, encoding thioredoxin reductase), confirming the mapping position of cydD previously established by P1-mediated transduction . Two ORFs identified by complementation experiments as cydD and cydC encode proteins with predicted molecular masses, respectively, of 65,103 and 62,946 Da . The hydropathy profile of each protein reveals an N-terminal hydrophobic domain and a C-terminal hydrophilic domain containing a putative nucleotide-binding site . The gene products probably constitute an ABC (ATP-binding cassette) family membrane transporter, the function of which is necessary for the formation of the cytochrome bd quinol oxidase . The CydDC system appears to be the first prokaryotic example of a heterodimeric ABC transport system in which each polypeptide contains both hydrophobic and ATP-binding domains.

EMBO J, 1993 Oct, 12(10), 3987 - 96
Formation of the central pseudoknot in 16S rRNA is essential for initiation of translation; Brink MF et al.; The postulated central pseudoknot formed by regions 9-13/21-25 and 17-19/916-918 of 16S rRNA of Escherichia coli is phylogenetically conserved in prokaryotic as well eukaryotic species . This pseudoknot is located at the center of the secondary structure of the 16S rRNA and connects the three major domains of this molecule . We have introduced mutations into this pseudoknot by changing the base-paired residues C18 and G917, and the effect of such mutations on the ribosomal activity was studied in vivo, using a 'specialized' ribosome system . As compared with ribosomes having the wild-type pseudoknot, the translational activity of ribosomes containing an A, G or U residue at position 18 was dramatically reduced, while the activity of mutant ribosomes having complementary bases at positions 18 and 917 was at the wild-type level . The reduced translational activity of those mutants that are incapable of forming a pseudoknot was caused by their inability to form 70S ribosomal complexes . These results demonstrate that the potential formation of a central pseudoknot in 16S rRNA with any base-paired residues at positions 18 and 917 is essential to complete the initiation process.

Biochim Biophys Acta, 1993 Sep 23, 1174(3), 241 - 57
cDNA clones contain autonomous replication activity; Wu C et al.; We have undertaken to investigate transcription as a regulatory event in mammalian DNA replication . Subpopulations of transcripts represented in a cDNA library of human embryo lung fibroblasts (IMR90) were examined for their ability to support autonomous replication after transfection into human cells (HeLa) . Two of three cDNA clones (343, 363) containing 'O'-family repetitive sequences, after subcloning into pBR322 and transfection into HeLa cells, were capable of autonomous replication . One of these cDNA clones, 343, is enriched by selection for poly(A)+ RNA . In contrast, none of five Alu-containing transcripts was capable of autonomous replication in human cells . However, six out of ten cDNA clones contained neither 'O'-family or Alu homologous sequences and were as efficient as the cDNA clones containing 'O'-family sequences in replicating autonomously in human cells . cDNA clones, from an oligo-d(T)-primed library of human poly(A)+ enriched RNA, contain a significant proportion of independent clones that can also support autonomous replication of bacterial plasmids in human cells . cDNA clone 343 was observed to contain in a 448 bp EcoRI-HincII fragment, yeast ARS consensus, SAR consensus, IRs, bent DNA and a DUE, all sequence and structural characteristics often associated with many prokaryotic, viral and eukaryotic origins . Sequence analysis of seven other cDNA clones (from non-'O'-family, non-Alu homologous sequences, NOA) showed that five contained some of the same consensus sequences . Two NOA clones (NOA4 and -5) did not contain any representations of ARS and SAR consensus sequences, suggesting that these two features may not be essential for autonomous replication activity in mammalian cells.

Biochemistry, 1993 Sep 21, 32(37), 9649 - 56
Cooperative binding of the Escherichia coli repressor of biotin biosynthesis to the biotin operator sequence; Abbott J et al.; Regulation of biotin biosynthesis and retention in Escherichia coli depends on a complex set of coupled protein-protein, protein-nucleic acid, and protein-small molecule interactions . The complexity of the biotin system is analogous to that found in gene regulatory systems from other prokaryotes and from eukaryotes . Quantitative understanding of these systems requires thermodynamic studies of the individual contributing interactions . We have initiated such studies of the biotin regulatory interactions . The assembly states of the biotin operon repressor (BirA) and its complex with the allosteric effector, bio-5'-AMP, have been determined by analytical gel filtration chromatography . Both the apo- and holo-repressors are monomeric at protein concentrations several orders of magnitude higher than those required for DNA binding . Results of stoichiometric DNA binding measurements indicate that the BirA-biotin operator (bioO) complex consists of two holo-repressor monomers per operator site . Equilibrium binding of BirA to bioO has been measured using the quantitative DNase footprint technique . Analysis of the data indicates that the binding process is best described by a cooperative model . An upper limit for the cooperative free energy is estimated to be between -2.0 and -3.0 kcal/mol.

J Mol Biol, 1993 Sep 20, 233(2), 219 - 30
Information analysis of sequences that bind the replication initiator RepA; Papp PP et al.; The replication initiator protein RepA of plasmid P1 can bind to 14 sites on the plasmid . These sites are variously used to autoregulate RepA synthesis and for initiation and control of DNA replication . Analysis of information (degree of conservation) at the sites revealed three sequence patches of high conservation . By saturation mutagenesis, the conservation at the outer two patches was found to contribute to RepA binding more critically . The guanine bases that are likely to contact RepA through the major groove were identified by methylation interference and methylation protection experiments . These bases mapped to the outer two patches and were separated by one turn of the helix . Therefore, they belong to major grooves on the same face of DNA . All backbone contacts of the protein, determined by hydroxyl radical footprinting, also mapped to the same face . We conclude from this that RepA binds to its site on one face of the DNA . Information analysis of binding sites for several prokaryotic repressors and activators, where the nature of DNA-protein contacts are known, revealed a correlation between the positions of high conservation and the positions of major grooves that faced the protein . The middle patch of high conservation in the RepA binding sites is an exception since in this region a minor groove is likely to face the protein . The simplest model for minor groove contacts suggests that in B-form DNA a T.A base-pair cannot easily be distinguished from an A.T pair by inspection of the minor groove . Yet in the RepA site, a T-->A mutation in the middle patch significantly affects binding . Therefore, the simplest models for both minor and major groove contacts are unlikely . It is possible that the patch determines the proper conformation of the site and thereby contributes to recognition indirectly.

Nature, 1993 Sep 16, 365(6443), 277 - 9
Structure of a new nucleic-acid-binding motif in eukaryotic transcriptional elongation factor TFIIS; Qian X et al.; Transcriptional elongation involves dynamic interactions among RNA polymerase and single-stranded and double-stranded nucleic acids in the ternary complex . In prokaryotes its regulation provides an important mechanism of genetic control . Analogous eukaryotic mechanisms are not well understood, but may control expression of proto-oncogenes and viruses, including the human immunodeficiency virus HIV-1 (ref . 8) . The highly conserved eukaryotic transcriptional elongation factor TFIIS enables RNA polymerase II (RNAPII) to read though pause or termination sites, nucleosomes and sequence-specific DNA-binding proteins . Two distinct domains of human TFIIS, which bind RNAPII and nucleic acids, regulate read-through and possibly nascent transcript cleavage . Here we describe the three-dimensional NMR structure of a Cys4 nucleic-acid-binding domain from human TFIIS . Unlike previously characterized zinc modules, which contain an alpha-helix, this structure consists of a three-stranded beta-sheet . Analogous Cys4 structural motifs may occur in other proteins involved in DNA or RNA transactions, including RNAPII itself . This new structure, designated the Zn ribbon, extends the repertoire of Zn-mediated peptide architectures and highlights the growing recognition of the beta-sheet as a motif of nucleic-acid recognition.

Gene, 1993 Sep 15, 131(2), 255 - 9
A phage T7 class-III promoter functions as a polymerase II promoter in mammalian cells; Sandig V et al.; A phage T7 class-III promoter (pT7), which is highly specific for T7 RNA polymerase in bacteria, was tested in mammalian cells for its specificity . After having shown that T7 RNA polymerase can transcribe from pT7 in the nucleus of stably transformed cells {Lieber et al., Nucleic Acids Res . 17 (1989) 8485-8493}, we describe here that pT7 could also direct efficient intracellular gene expression in the absence of T7 RNA polymerase . Using the genomic human growth hormone-encoding gene and the firefly luciferase-encoding gene as reporters, we found expression levels comparable with those obtained with the Rous sarcoma viral promoter . Inhibition of expression with alpha-amanitin suggests that transcription is by RNA polymerase II . Binding studies with HeLa cell extracts clearly show that synthetic pT7 sequences are specifically bound (gel retardation) and that the promoter region is protected from DNase degradation . The experimental data, as well as the nucleotide sequence, suggest that pT7 has properties of an initiator element . Indeed, the activity of pT7 can be stimulated by the presence of an upstream element or an enhancer . These results have practical implications for the use of pT7 in mammalian expression vectors . Commercial pT7 plasmids can be used for both prokaryotic and eukaryotic expression systems.

Experientia, 1993 Sep 15, 49(9), 825 - 9
The structure of scytonemin, an ultraviolet sunscreen pigment from the sheaths of cyanobacteria; Proteau PJ et al.; Despite knowledge of the existence of the pigment called scytonemin for over 100 years, its structure has remained unsolved until now . This pigment, the first shown to be an effective, photo-stable ultraviolet shield in prokaryotes, is a novel dimeric molecule (molec . wt . 544) of indolic and phenolic subunits and is known only from the sheaths enclosing the cells of cyanobacteria . It is probable that scytonemin is formed from a condensation of tryptophan- and phenylpropanoid-derived subunits . The linkage between these units is unique among natural products and this novel ring structure is here termed the 'scytoneman skeleton' . Scytonemin absorbs strongly and broadly in the spectral region 325-425 nm (UV-A-violet-blue, with an in vivo maximum at 370 nm) . However, there is also major absorption in the UV-C (lambda max = 250 nm) and UV-B (280-320 nm) . The pigment has been recently shown to provide significant protection to cyanobacteria against damage by ultraviolet radiation . The pigment occurs in all phylogenetic lines of sheathed cyanobacteria and possibly represents a UV screening strategy far more ancient than that of plant flavonoids and animal melanins . How diverse organisms deal with UV radiation is considered of vital importance to global ecology.

J Immunol Methods, 1993 Sep 15, 164(2), 221 - 31
Recombinant human preproinsulin . Expression, purification and reaction with insulin autoantibodies in sera from patients with insulin-dependent diabetes mellitus; Berg H et al.; A novel prokaryotic expression vector pGEX-6T was designed for high-level expression of recombinant fusion protein with a histidine-hexapeptide and glutathione-S-transferase at its N-terminus and the recombinant human preproinsulin at its C-terminus . Efficiency of expression was investigated in the Escherichia coli strain CAG456 . The synthesized protein was sequestered in an insoluble form in inclusion bodies and was purified to homogeneity by one-step affinity chromatography based on the specific complex formation of the histidine-hexapeptide and a chelating matrix which was charged with Ni2+ ions . The antigenic nature of the purified recombinant preproinsulin fusion protein was evaluated by ELISA screening for insulin autoantibodies in selected sera from patients with recent-onset type 1 (insulin-dependent) diabetes mellitus classified by the existence of additional autoantibodies reactive against glutamic acid decarboxylase . 14% of the tested sera (n = 43) contained insulin autoantibodies which strongly recognized the recombinant human preproinsulin . Comparable measurements with both recombinant human preproinsulin and mature insulin suggested that the observed autoantigenicity of preproinsulin was mediated by the C-peptide or/and signal peptide.

Nucleic Acids Res, 1993 Sep 11, 21(18), 4268 - 71
Influence of the three nucleotides upstream of the initiation codon on expression of the Escherichia coli lacZ gene in Saccharomyces cerevisiae; Looman AC et al.; By introducing synthetic oligonucleotides into a lacZ-yeast expression vector a set of 47 plasmids (out of 64 possible) was generated, differing only in the three bases immediately upstream of the AUG initiation codon of the Escherichia coli lacZ gene . Expression of the beta-galactosidase fusion protein encoded by the different plasmids was determined in Saccharomyces cerevisiae by immunogel electrophoresis . Among the clones tested we found a factor 3 difference in expression . A slight nucleotide preference was found in positions -3(A > G > C = U) and -2 (G > C = U > A) . The choice of the nucleotide at position -1 immediately 5' of the AUG did not effect translation efficiency . Increasing homology to the yeast consensus sequence (AAAAAAAUGUCU) was not concomitant with an increased translation efficiency . Our results indicate that the choice of nucleotides immediately preceding the initiation codon in yeast does not dramatically influence translation efficiency, as in prokaryotes or higher eukaryotes.

Nucleic Acids Res, 1993 Sep 11, 21(18), 4200 - 5
Mutations dissociating the inhibitory activity of the pokeweed antiviral protein on eukaryote translation and Escherichia coli growth; Dore JM et al.; The pokeweed antiviral protein is a ribosome inactivating protein acting on eukaryotic as well as on prokaryotic ribosomes thus is toxic for both cell types . Using the PCR technique to clone the PAP open reading frame, we characterized two cDNAs coding for proteins inhibiting eukaryotic translation process and which are not toxic for Escherichia coli, unlike the wild type protein . The sequence of the two cDNAs showed that the proteins contain only one and two point mutations . This result suggest that the wild type amino acids in the mutated positions participate in the prokaryotic ribosome recognition . These mutants might be useful for the construction of immunotoxins containing the pokeweed antiviral protein as toxin.

FEBS Lett, 1993 Sep 6, 330(1), 99 - 104
The cyanobacterium, Synechococcus sp . PCC7942, possesses two distinct genes encoding cation-transporting P-type ATPases; Kanamaru K et al.; P-type (or E1 E2-type) ATPases comprise a large family of prokaryotic and eukaryotic proteins capable of transporting a variety of cations, and function in a wide variety of cellular processes . The present study was carried out to search for genes encoding P-type ATPases in the phototrophic cyanobacterium, Synechococcus sp . PCC7942 . We succeeded in cloning two genes each encoding P-type ATPases from this bacterium . It was found that Synechococcus at least, two distinct P-type ATPases; one belongs to the family of typical prokaryotic P-type ATPases and the other markedly resembles eukaryotic P-type ATPases . An insertion mutant lacking either of these two ATPase-genes was constructed . The results showed that the growth of these mutants is hypersensitive to osmotic stress upon addition of NaCl or sorbitol to the medium.

J Biol Chem, 1993 Sep 5, 268(25), 18567 - 72
Identification of glutamic acid 204 and aspartic acid 200 in chitinase A1 of Bacillus circulans WL-12 as essential residues for chitinase activity; Watanabe T et al.; Prokaryotic chitinases, class III plant chitinases, yeast chitinases, and endo-beta-N-acetylglucosaminidases share weak amino acid sequence similarities at the certain region of each enzyme . These regions have been assumed to be important for catalytic activities of the enzymes . To verify this assumption, three amino acid residues (Ser-160, Asp-200, Glu-204) in chitinase A1 of Bacillus circulans WL-12 were chosen, based on the amino acid sequence alignment of the regions sharing sequence similarity, and were replaced by site-directed mutagenesis . Kinetic parameters for 4-methylumbelliferyl-N,N',N"-triacetylchitotriose hydrolysis were determined with wild-type and seven mutant chitinases . Chitinases with Glu-204-->Gln mutation and Glu-204-->Asp mutation were essentially inactive and kcat values of these chitinases were approximately 1/5,000 and 1/17,000 of that of wild-type chitinase, respectively . Asp-200-->Asn mutation decreased the kcat value to approximately 1/350 of that of the wild-type enzyme, while the Km value decreased only slightly . On the other hand, neither the kcat value nor the Km value was affected by Asp-200-->Glu mutation . Thus, it appeared that Glu-204 and Asp-200 are directly involved in the catalytic events of chitinase A1 . The role of the carboxyl group of Asp-200 can be fully substituted by that of Glu residue . The Ser-160-->Ala mutant retained 10% activity of the wild-type chitinase indicating that the hydroxyl group of Ser-160 is not absolutely required for the catalytic activity . These results indicate a lysozyme-type catalytic mechanism of the chitinase.

Mol Gen Genet, 1993 Sep, 240(3), 445 - 9
Ribosomal protein gene rpl5 is cotranscribed with the nad3 gene in Oenothera mitochondria; Schuster W; The rpl5 ribosomal protein gene was identified in the mitochondrial genome of the higher plant Oenothera berteriana . The gene is present in a unique genomic location upstream of the gene encoding subunit 3 of the NADH dehydrogenase (nad3) . Both genes are cotranscribed, and the mRNA is modified at several cytidine residues by RNA editing . Analysis of the editing profiles of both genes by direct cDNA analysis and polymerase chain reaction (PCR) revealed that not all transcripts are fully edited at all sites . Eight of the nine C to U conversions in the rpl5 reading frame are non-silent and change the deduced amino acid sequence . The genes of the prokaryotic-like cistron that includes the rpsl9, rps3, rpl16, rpl5, and rpsl4 genes, which is at least partially conserved in the mitochondrial genomes of other higher and lower plants, are dispersed in the Oenothera mitochondrial genome.

J Hered, 1993 Sep-Oct, 84(5), 351 - 9
The sexual nature of the eukaryote genome; Bell G; This paper supports a previous conjecture that the sexual cycle of eukaryotes arose from the infection of cells by genome parasites . The finding are as follows . (1) In prokaryotes, conjugative plasmids ensure their own spread by directing partial cell fusion . (2) Conjugative plasmids permit gene transfer between prokaryotes and eukaryotes, and can be integrated into the eukaryote genome . (3) Genes can be transferred between unrelated eukaryotes, and between different genomes within eukaryotic cells . (4) Elements such as transposons and retroviruses evolve as parasites of the eukaryote sexual system . (5) The mating-type genes of bipolar fungi are idiomorphic: alternative genes directing sexual specificity are dissimilar and non-homologous . It is argued that they arose as parasitic elements directing cell fusion, which have become integrated into the genome . (6) Mating-type idiomorphs in different taxa may be dissimilar, reflecting the independent acquisition of different infectious elements by different eukaryote lineages . (7) Sexual competence is rapidly lost through mutation accumulation or antagonistic pleiotropy during vegetative proliferation, so that many lineages are sexually sterile . (8) In multipolar systems, novel mating-type alleles arising by mutation spread in a parasite-like manner by virtue of their access to lineages which have become sexually sterile . (9) It is suggested that centromeres arise as devices to enable low copy number plasmids to persist . (10) Crossing over is favored if it enables the genes that direct it to unlink themselves from inferior genomes, or from the consequences of their own transposition.(ABSTRACT TRUNCATED AT 250 WORDS)

J Clin Microbiol, 1993 Sep, 31(9), 2502 - 5
Typing of Aspergillus species and Aspergillus fumigatus isolates by interrepeat polymerase chain reaction; van Belkum A et al.; Polymerase chain reaction amplification of repetitive DNA motifs allows discrimination of Aspergillus species . In a preliminary survey, the DNA fingerprints appear to be identical when isolates of Aspergillus fumigatus are compared . When a primer deduced from a prokaryotic repeat motif is used, Aspergillus fumigatus isolates originating from different patients or different anatomical locations can be typed individually.

Comp Biochem Physiol B, 1993 Sep, 106(1), 1 - 26
Adventitious variability? The amino acid sequences of nonvertebrate globins; Vinogradov SN et al.; 1 . The more than 140 amino acid sequences of non-vertebrate hemoglobins (Hbs) and myoglobins (Mbs) that are known at present, can be divided into several distinct groups: (1) single-chain globins, containing one heme-binding domain; (2) truncated, single-chain, one-domain globins; (3) chimeric, one-domain globins; (4) chimeric, two-domain globins; and (5) chimeric multi-domain globins . 2 . The crystal structures of eight nonvertebrate Hbs and Mbs are known, all of them monomeric, one-domain globin chains . Although these molecules represent plants, prokaryotes and several metazoan groups, and although the inter-subunit interactions in the dimeric and tetrameric molecules differ from the ones observed in vertebrate Hbs, the secondary structures of all seven one-domain globins retain the characteristic vertebrate "myoglobin fold" . No crystal structures of globins representing the other four groups have been determined . 3 . Furthermore, a number of the one-, two- and multi-domain globin chains participate in a broad variety of quaternary structures, ranging from homo- and heterodimers to highly complex, multisubunit aggregates with M(r) > 3000 kDa (S . N . Vinogradov, Comp . Biochem . Physiol . 82B, 1-15, 1985) . 4 . (1) The single-chain, single-domain globins are comparable in size to the vertebrate globins and exhibit the widest distribution . (A) Intracellular Hbs include: (i) the monomeric and polymeric Hbs of the polychaete Glycera; (ii) the tetrameric Hb of the echiuran Urechis; (iii) the dimeric Hbs of echinoderms such as Paracaudina and Caudina; and (iv) the dimeric and tetrameric Hbs of molluscs, the bivalves Scapharca, Anadara, Barbatia and Calyptogena . (B) Extracellular Hbs include: (i) the multiple monomeric and dimeric Hbs of the larva of the insect Chironomus; (ii) the Hbs of nematodes such as Trichostrongylus and Caenorhabditis; (iii) the globin chains forming tetramers and dodecamers and comprising approximately 2/3 of the giant (approximately 3600 kDa), hexagonal bilayer (HBL) Hbs of annelids, e.g . the oligochaete Lumbricus and the polychaete Tylorrhynchus and of the vestimentiferan Lamellibrachia; and (iv) the globin chains comprising the ca 400 kDa Hbs of Lamellibrachia and the pogonophoran Oligobrachia . (C) Cytoplasmic Hbs include: (i) the Mbs of molluscs, the gastropods Aplysia, Bursatella, Cerithedea, Nassa and Dolabella and the chiton Liolophura; (ii) the three Hb of the symbiont-harboring bivalve Lucina; (iii) the dimeric Hb of the bacterium Vitreoscilla; and (iv) plant Hbs, including the Hbs of symbiont-containing legumes (Lgbs), the Hbs of symbiont-containing non-leguminous plants and the Hbs in the roots of symbiont-free plants.(ABSTRACT TRUNCATED AT 400 WORDS)

Mol Cell Biol, 1993 Sep, 13(9), 5315 - 22
Inverted DNA repeats: a source of eukaryotic genomic instability; Gordenin DA et al.; While inverted DNA repeats are generally acknowledged to be an important source of genetic instability in prokaryotes, relatively little is known about their effects in eukaryotes . Using bacterial transposon Tn5 and its derivatives, we demonstrate that long inverted repeats also cause genetic instability leading to deletion in the yeast Saccharomyces cerevisiae . Furthermore, they induce homologous recombination . Replication plays a major role in the deletion formation . Deletions are stimulated by a mutation in the DNA polymerase delta gene (pol3) . The majority of deletions result from imprecise excision between small (4- to 6-bp) repeats in a polar fashion, and they often generate quasipalindrome structures that subsequently may be highly unstable . Breakpoints are clustered near the ends of the long inverted repeats (< 150 bp) . The repeats have both intra- and interchromosomal effects in that they also create hot spots for mitotic interchromosomal recombination . Intragenic recombination is 4 to 18 times more frequent for heteroalleles in which one of the two mutations is due to the insertion of a long inverted repeat, compared with other pairs of heteroalleles in which neither mutation has a long repeat . We propose that both deletion and recombination are the result of altered replication at the basal part of the stem formed by the inverted repeats.

Proc Natl Acad Sci U S A, 1993 Sep 1, 90(17), 8140 - 4
A transcription inhibitor specific for unwound DNA in RNA polymerase-promoter open complexes; Mazumder A et al.; The kinetically component open complexes formed at prokaryotic and eukaryotic transcription start sites are efficiently nicked by the chemical nuclease activity of the 2:1 1,10-phenanthroline-copper(I) complex {(OP)2Cu+} and hydrogen peroxide . This reaction specificity has been attributed to the creation of a binding site(s) for redox-active tetrahedral (OP)2Cu+ when RNA polymerase form productive complexes with promoters . This proposal has been confirmed for the Escherichia coli lac UV-5 promoter by the demonstration that the 2:1 2,9-dimethyl-1,10-phenanthroline-copper(I) complex {(Me2OP)2Cu+}, a redox-inactive isostere of (OP)2-Cu+, protects the transcription start site from scission by the chemical nuclease activity . (Me2OP)2Cu+ is also an effective inhibitor of transcription . The inhibition of transcription and the protection from scission of the open complex by (OP)2Cu+ exhibit the same dependence on the concentration of (Me2OP)2Cu+ . This redox- and exchange-stable species is a previously undescribed transcription inhibitor that binds to a site generated by the interaction of RNA polymerase with the promoter . Unlike the intercalating agent proflavine, which is also an effective transcription inhibitor, it does not displace the enzyme from the promoter . The ability of (Me2OP)2Cu+ to inhibit transcription may be partially responsible for its potent cytotoxicity.

J Bacteriol, 1993 Sep, 175(17), 5666 - 76
The major iron-containing protein of Legionella pneumophila is an aconitase homologous with the human iron-responsive element-binding protein; Mengaud JM et al.; Legionella pneumophila has high iron requirements, and its intracellular growth in human monocytes is dependent on the availability of intracellular iron . To learn more about iron metabolism in L . pneumophila, we have undertaken an analysis of the iron proteins of the bacterium . We first developed an assay to identify proteins by 59Fe labelling and nondenaturing polyacrylamide gel electrophoresis . The assay revealed seven iron proteins (IPs) with apparent molecular weights of 500, 450, 250, 210, 150, 130, and 85 . IP150 comigrates with superoxide dismutase activity and is probably the Fe-superoxide dismutase of L . pneumophila . IP210 is the major iron-containing protein (MICP) . To identify and characterize MICP, we purified the protein and cloned and sequenced its gene . MICP is a monomeric protein containing 891 amino acids, and it has a calculated molecular mass of 98,147 Da . Analysis of the sequence revealed that MICP has two interesting homologies . First, MICP is highly homologous with the human iron-responsive element-binding protein, consistent with the hypothesis that this critical iron-regulatory molecule of humans has a prokaryotic ancestor . Second, MICP is highly homologous with the Escherichia coli aconitase and to a lesser extent with porcine heart mitochondrial aconitase . Consistent with this, we found that MICP exhibits aconitase activity . In contrast to other aconitases, MICP has a single amino acid change of a potentially deleterious type at a site thought to be critical for substrate binding and enzymatic activity . However, the specific activity of MICP is roughly comparable to that of other aconitases, suggesting that the mutation has at most a mild effect on the aconitase activity of MICP . The abundance of MICP in L . pneumophila suggests either that L . pneumophila requires high aconitase and perhaps tricarboxylic acid cycle activity or that the bacterium requires large amounts of this protein to serve an additional role in bacterial physiology . A need for large amounts of MICP, which contains four Fe atoms per molecule when fully loaded, could at least partly explain L . pneumophila's high metabolic requirement for iron.

J Bacteriol, 1993 Sep, 175(17), 5548 - 58
Cloning, primary structure, and regulation of the HIS7 gene encoding a bifunctional glutamine amidotransferase: cyclase from Saccharomyces cerevisiae; Kuenzler M et al.; The Saccharomyces cerevisiae HIS7 gene was cloned by its location immediately downstream of the previously isolated and characterized ARO4 gene . The two genes have the same orientation with a distance of only 416 bp between the two open reading frames . The yeast HIS7 gene represents the first isolated eukaryotic gene encoding the enzymatic activities which catalyze the fifth and sixth step in histidine biosynthesis . The open reading frame of the HIS7 gene has a length of 1,656 bp resulting in a gene product of 552 amino acids with a calculated molecular weight of 61,082 . Two findings implicate a bifunctional nature of the HIS7 gene product . First, the N-terminal and C-terminal segments of the deduced HIS7 amino acid sequence show significant homology to prokaryotic monofunctional glutamine amidotransferases and cyclases, respectively, involved in histidine biosynthesis . Second, the yeast HIS7 gene is able to suppress His auxotrophy of corresponding Escherichia coli hisH and hisF mutants . HIS7 gene expression is regulated by the general control system of amino acid biosynthesis . GCN4-dependent and GCN4-independent (basal) transcription use different initiator elements in the HIS7 promoter.

Virology, 1993 Sep, 196(1), 89 - 100
Alterations in cell membrane permeability by the lentivirus lytic peptide (LLP-1) of HIV-1 transmembrane protein; Miller MA et al.; Previously we reported that synthetic peptide homologs of an amphipathic region (designated the lentivirus lytic peptide, or LLP-1) near the carboxy terminus of HIV-1 transmembrane protein (TM) were toxic for both prokaryotic and eukaryotic cells when added exogenously to cell cultures . We postulated that these peptides may exert their toxic effects in much the same manner as natural cytolytic peptides such as magainins, cecropins, and melittin by forming pores through cellular membranes . Here we show the results of 51Cr-release assays and membrane flux measurements of peptide treated cells that support our hypothesis . We have also tested a limited panel of LLP-1 peptide analogs in these assays and found that relatively minor alterations in peptide charge or amphipathicity in the parent HIV LLP-1 sequence resulted in total loss of membrane perturbative properties . These results demonstrate that the peptide homolog of HIV-1 LLP-1 can indeed perturb membranes by forming pores of defined size in cytoplasmic membranes . Furthermore, the analog studies described here reveal that the amphipathy and high positive charge of this protein segment are required for the membrane perturbative properties.

Mol Cell Biol, 1993 Sep, 13(9), 5370 - 6
Identification of residues in the human DNA repair enzyme HAP1 (Ref-1) that are essential for redox regulation of Jun DNA binding; Walker LJ et al.; The DNA binding activity of the c-jun proto-oncogene product is inhibited by oxidation of a specific cysteine residue (Cys-252) in the DNA binding domain . Jun protein inactivated by oxidation of this residue can be efficiently reactivated by a factor from human cell nuclei, recently identified as a DNA repair enzyme (termed HAP1 or Ref-1) . The HAP1 protein consists of a core domain, which is highly conserved in a family of prokaryotic and eukaryotic DNA repair enzymes, and a 61-amino-acid N-terminal domain absent from bacterial homologs such as Escherichia coli exonuclease III . The eukaryote-specific N-terminal domain was dispensable for the DNA repair functions of the HAP1 protein but was essential for reactivation of the DNA binding activity of oxidized Jun protein . Consistent with this finding, exonuclease III protein could not reactive Jun . A minimal 26-residue region of the N-terminal domain proximal to the core of the HAP1 enzyme was required for redox activity . By site-directed mutagenesis, cysteine 65 was identified as the redox active site in the HAP1 enzyme . In addition, it is proposed that cysteine 93 interacts with the redox active site, probably via disulfide bridge formation . It is concluded that the HAP1 protein has evolved a novel redox activation domain capable of regulating the DNA binding activity of a proto-oncogene product which is not essential for its DNA repair functions . Identification of a putative active site cysteine residue should facilitate analysis of the mechanism by which the HAP1 protein may alter the redox state of a wide range of transcription factors.

J Biochem (Tokyo), 1993 Sep, 114(3), 350 - 7
Novel members of the two-component signal transduction genes in Escherichia coli; Nagasawa S et al.; A variety of adaptive response systems in prokaryotes often involve two families (two components) of signal transduction proteins, namely, sensory kinases and response-regulators . To extend the list of such sensor/regulator genes for Escherichia coli, we adopted a random screening method in this study . In particular, we isolated a series of recombinant plasmids that are able phenotypically to suppress mutational lesions of both the envZ and phoR/creC genes, each of which encodes a well-characterized sensory-kinase . Among the recombinant plasmids thus isolated, two clones (named pSN11 and pSN25) were subjected to characterization in detail . These analyses allowed us to identify the genetic loci specifying novel members of the sensor/regulator families . One (pSN11) is located around 45 min on the E . coli genetic map, that contains two adjacent coding-sequences (baeS and baeR) . The other (pSN25) is located around 93 min of the genetic map, that also comprises two adjacent coding-sequences (basS and basR) . These two pairs of gene-products, thus newly identified, were revealed to belong to typical members of the sensor/regulator families . Furthermore, they were demonstrated to exhibit the in vitro phosphotransfer reaction in the presence of ATP, that is also a characteristic of the sensory-kinase and response-regulator proteins.

EMBO J, 1993 Sep, 12(9), 3703 - 8
Phage P4 alpha protein is multifunctional with origin recognition, helicase and primase activities; Ziegelin G et al.; alpha Protein of satellite phage P4 of Escherichia coli is multifunctional in P4 replication with three activities . First, the protein (subunit M(r) = 84,900) complexes specifically the P4 origin and the cis replication region required for replication . alpha Protein interacts with all six type I repeats (TGTTCACC) present in the origin . Second, associated with the alpha protein is a DNA helicase activity that is fueled by hydrolysis of a nucleoside 5' triphosphate . All common NTPs except UTP and dTTP can serve as cofactors . Strand separation of partial duplexes containing tailed ends that resemble a replication fork is preferred, although a preformed fork is not absolutely required for the enzyme to invade and unwind duplex DNA . alpha Protein catalyzes unwinding in the 3'-5' direction with respect to the strand it has bound . Finally, the primase activity already demonstrated for alpha protein is due to synthesis of RNA primers . In vitro, alpha protein generates di- to pentaribonucleotides on single-stranded phage fd DNA . The predominant product is the dimer pppApG, on which most of the longer oligoribonucleotides are based . Using DNA oligonucleotides of defined sequence as templates, synthesis of pppApG was also detectable . To date, among prokaryotic and eukaryotic replication systems, gp alpha is the only protein known that combines three activities on one single polypeptide chain.

EMBO J, 1993 Sep, 12(9), 3373 - 83
Bradyrhizobium japonicum TlpA, a novel membrane-anchored thioredoxin-like protein involved in the biogenesis of cytochrome aa3 and development of symbiosis; Loferer H et al.; We report the discovery of a bacterial gene, tlpA, that codes for a hitherto unknown type of thioredoxin-like protein . The gene was found in the course of studying a Tn5 insertion mutant of the soybean root nodule symbiont Bradyrhizobium japonicum . The TlpA protein shared up to 31% amino acid sequence identity with various eukaryotic and prokaryotic thioredoxins and protein disulfide isomerases, and possessed a characteristic active-site sequence, Trp-Cys-Val-Pro-Cys . In contrast to all members of the thioredoxin family known to date, TlpA was shown to be anchored to the cytoplasmic membrane by means of an N-terminal transmembrane domain, while the active site-containing part of the protein faced the periplasm . The tlpA mutant had a pleiotropic phenotype in that it was defective in the development of a nitrogen fixing endosymbiosis and exhibited a strongly decreased oxidase activity, as compared with the wild-type . Holocytochrome aa3 was spectroscopically undetectable in the mutant, whereas the apoprotein of subunit one (CoxA) of this oxidase was still synthesized and incorporated into the cytoplasmic membrane . Since cytochrome aa3 is not a prerequisite for the development of symbiosis, the results suggest that TlpA is involved in at least two independent cellular processes, one of which is an essential periplasmic step in the maturation of cytochrome aa3.

Microbiol Rev, 1993 Sep, 57(3), 703 - 24
Genetics of eukaryotic RNA polymerases I, II, and III; Archambault J et al.; The transcription of nucleus-encoded genes in eukaryotes is performed by three distinct RNA polymerases termed I, II, and III, each of which is a complex enzyme composed of more than 10 subunits . The isolation of genes encoding subunits of eukaryotic RNA polymerases from a wide spectrum of organisms has confirmed previous biochemical and immunological data indicating that all three enzymes are closely related in structures that have been conserved in evolution . Each RNA polymerase is an enzyme complex composed of two large subunits that are homologous to the two largest subunits of prokaryotic RNA polymerases and are associated with smaller polypeptides, some of which are common to two or to all three eukaryotic enzymes . This remarkable conservation of structure most probably underlies a conservation of function and emphasizes the likelihood that information gained from the study of RNA polymerases from one organism will be applicable to others . The recent isolation of many mutations affecting the structure and/or function of eukaryotic and prokaryotic RNA polymerases now makes it feasible to begin integrating genetic and biochemical information from various species in order to develop a picture of these enzymes . The picture of eukaryotic RNA polymerases depicted in this article emphasizes the role(s) of different polypeptide regions in interaction with other subunits, cofactors, substrates, inhibitors, or accessory transcription factors, as well as the requirement for these interactions in transcription initiation, elongation, pausing, termination, and/or enzyme assembly . Most mutations described here have been isolated in eukaryotic organisms that have well-developed experimental genetic systems as well as amenable biochemistry, such as Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans . When relevant, mutations affecting regions of Escherichia coli RNA polymerase that are conserved among eukaryotes and prokaryotes are also presented . In addition to providing information about the structure and function of eukaryotic RNA polymerases, the study of mutations and of the pleiotropic phenotypes they imposed has underscored the central role played by these enzymes in many fundamental processes such as development and cellular differentiation.

J Biolumin Chemilumin, 1993 Sep-Oct, 8(5), 267 - 91
Bioluminescence and chemiluminescence literature . Luciferase reporter genes--lux and luc . Part 2; Hill PJ et al.; The following references encompass a review of recent literature where the in vivo expression of eukaryotic and/or prokaryotic luciferases provide for sensitive reporters of cellular activity . The list is subdivided into prokaryotic and eukaryotic applications . We have included the uses of luciferases in elucidating the control of gene expression or for monitoring cell viability . We have not included papers cited by Stanley and Stewart (J Biolumin Chemilumin 1990; 5:141-52) nor have we included papers on the structure and regulation of luciferases as this now substantial literature will be the subject of a future review . References cited in both this review and previous ones are referred to by the number assigned to them in the earlier review.

Curr Genet, 1993 Sep, 24(3), 256 - 9
The presequence of cytochrome c1 from potato mitochondria is encoded on four exons; Wegener S et al.; The structural organization of a nuclear gene encoding cytochrome c1 from potato was determined . The gene spans 5.1 kb and contains eight introns . All intron/exon junctions follow the GT/AG rule . Functional domains of the mature cytochrome c1 protein are located on separate exons . The presequence, which targets the cytochrome c1 precursor to the mitochondrion and to the correct intra-mitochondrial location, is encoded on the first four exons . The largest intron (2.8 kb) separates the information for mitochondrial targeting from the "intra-mitochondrial sorting domain" of the cytochrome c1 protein . In contrast to other organellar precursor proteins, there is no intron between the DNA sequence encoding the presequence and the mature protein . This may indicate that during evolution the genetic information for the prokaryotic cytochrome c1 was transferred to the nucleus together with the bacterial secretion signal which is structurally and functionally related to "intramitochondrial sorting domains".

Trends Microbiol, 1993 Sep, 1(6), 236 - 9
Linear DNA of Borrelia species and antigenic variation; Barbour AG; Members of the genus Borrelia may be unique among prokaryotic organisms in having a polyploid genome that is mostly linear . The smaller linear duplex replicons in these organisms have been called plasmids, but there is justification for designating them minichromosomes instead . The antigenic identities of the agents of Lyme disease and relapsing fever are largely determined by these extrachromosomal genes.

Protein Sci, 1993 Sep, 2(9), 1490 - 6
Prolyl isomerases catalyze antibody folding in vitro; Lilie H et al.; Some slow-folding phases in the in vitro refolding of proteins originate from the isomerization of prolyl-peptide bonds, which can be accelerated by a class of enzymes called prolyl isomerases (PPIs) . We used the in vitro folding of an antibody Fab fragment as a model system to study the effect of PPI on a folding reaction that is only partially reversible . We show here that members of both subclasses of PPIs, cyclophilin and FK 506 binding protein (FKBP), accelerate the refolding process and increase the yield of correctly folded molecules . An acceleration of folding was not observed in the presence of the specific inhibitor cyclosporin A, but still the yield of correctly folded molecules was increased . Bovine serum albumin (BSA) increased the yield comparable to cyclophilin but, in contrast, did not influence the rate of reactivation . These effects were observed only when cyclophilin or BSA were present during the first few seconds of refolding . However, the rate-limiting reactivation reaction is still accelerated when PPI is added several minutes after starting refolding . In contrast, the prokaryotic chaperone GroEL influences the refolding yield when added several minutes after initiating refolding . The results show that PPIs influence the folding of Fab in two different ways . (1) They act as true catalysts of protein folding by accelerating the rate-limiting isomerization of Xaa-Pro peptide bonds . Proline isomerization is obviously a late folding step and has no influence on the formation of aggregates within the first seconds of the refolding reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Microbiol, 1993 Sep, 9(6), 1283 - 95
The IS4 family of insertion sequences: evidence for a conserved transposase motif; Rezsohazy R et al.; The eight IS231 variants characterized so far (IS231 A-F, V and W) display similar transposases with an overall 40% identity . Comparison with all the prokaryotic transposable elements sequenced so far revealed that the IS231 transposases share two conserved regions with those of 35 other insertion sequences of wide origins . These insertion sequences, defining the IS4 family, have a common bipartite organization of their ends and are divided into two similarity groups . Interestingly, the transposase domains conserved within this family display similarities with the well known integrase domain shared by transposases of the IS3 and IS15 families, and integrases of retroelements . This domain is also found in IS30-related elements and Tn7 TnsB protein . Amino acid residues conserved throughout all these prokaryotic and eukaryotic mobile genetic elements define a major transposase/integrase motif, likely to play an important role in the transposition process.

Mol Microbiol, 1993 Sep, 9(5), 989 - 98
The catR gene encoding a catalase from Aspergillus niger: primary structure and elevated expression through increased gene copy number and use of a strong promoter; Fowler T et al.; Synthetic oligonucleotide probes based on amino acid sequence data were used to identify and clone cDNA sequences encoding a catalase (catalase-R) of Aspergillus niger . One cDNA clone was subsequently used to isolate the corresponding genomic DNA sequences (designated catR) . Nucleotide sequence analysis of both genomic and cDNA clones suggested that the catR coding region consists of five exons interrupted by four small introns . The deduced amino acid sequence of catalase-R spans 730 residues which show significant homology to both prokaryotic and eukaryotic catalases, particularly in regions involved in catalytic activity and binding of the haem prosthetic group . Increased expression of the catR gene was obtained by transformation of an A . niger host strain with an integrative vector carrying the cloned genomic DNA segment . Several of these transformants produced three- to fivefold higher levels of catalase than the untransformed parent strain . Hybridization analyses indicated that these strains contained multiple copies of catR integrated into the genome . A second expression vector was constructed in which the catR coding region was functionally joined to the promoter and terminator elements of the A . niger glucoamylase (glaA) gene . A . niger transformants containing this vector produced from three- to 10-fold higher levels of catalase-R than the untransformed parent strain.

Eur J Biochem, 1993 Sep 1, 216(2), 607 - 13
Structure and metabolic control of the Yarrowia lipolytica peroxisomal 3-oxoacyl-CoA-thiolase gene; Berninger G et al.; Using a Yarrowia lipolytica genomic library, several overlapping clones of the peroxisomal 3-oxoacyl-CoA-thiolase gene, POT1, were isolated . The library was prepared in the bacterial expression vector lambda gt11, thus allowing an immunological screening of recombinant bacteriophages with specific antibodies raised against purified peroxisomal thiolase . The isolated POT1 clones hybridized to a 1.4 kb RNA species, which was induced approximately 30-fold when oleate was the carbon source . A 3634-bp segment of the cloned DNA was sequenced . This segment contained, on both strands, three major overlapping open-reading frames of 678, 1122 and 1242 bp . Northern-hybridization analysis showed that only the largest of these reading frames was transcribed . It encodes a protein of 414 amino acids and molecular mass 43.059 kDa . Its deduced amino acid sequence has 30-60% identity and 50-70% sequence similarity when compared to other known thiolases . According to both the amount (68-71%) and location of conserved amino acids, the encoded protein belongs to the peroxisomal rather than the mitochondrial or cytoplasmic class of thiolases . Compared to bacterial and yeast cytosolic thiolases, the POT1 gene product contains a N-terminal extension of 25 amino acids which clearly differs from typical mitochondrial import signals . One of the isolated clones contained, in addition to the POT1 coding sequence, 784 bp of the corresponding 5' flanking region . Nevertheless, it was efficiently expressed in Escherichia coli suggesting the correct recognition of this fungal promoter by the prokaryotic transcriptional and translational machinery . The Y . lipolytica genomic POT1 gene was disrupted by replacing 120 bp of its coding sequence with 2.7 kbp of DNA including the Y . lipolytica LEU2 gene . The resulting delta pot1::LEU2 cells were free of immunologically cross-reacting thiolase . Western-blot analysis showed that the product of the non-disrupted gene had a molecular mass of approximately 42 kDa . This corresponds well to the molecular mass of purified Y . lipolytica peroxisomal thiolase . Disruption of POT1 abolished the ability of Y . lipolytica cells to grow on solid media with oleate as a carbon source . This inability to grow in the presence of oleate suggests both the catabolic function of POT1 and the absence of additional catabolic thiolases in Y . lipolytica . However, the delta pot1::LEU2 cells were unaffected in their ability to elongate externally added tridecanoic acid to its higher-chain-length homologues . Hence, another, POT1-independent and biosynthetic 3-oxoacyl-CoA thiolase must be responsible for this reaction in Y . lipolytica.

Trends Neurosci, 1993 Sep, 16(9), 365 - 70
The elusive transporters with a high affinity for glutamate; Kanai Y et al.; Removal of glutamate from the synaptic cleft is an essential component of the transmission process at glutamatergic synapses . This requirement is fulfilled by transporters that have a high affinity for glutamate and exhibit a unique coupling to Na+, K+ and OH- ions . Independently, three groups have succeeded in cloning cDNAs encoding high-affinity Na(+)-dependent glutamate transporters . These transporters are structurally distinct from previously characterized neurotransmitter transporters and show sequence identity with prokaryotic glutamate and dicarboxylate transporters . In addition, they exhibit significant differences in their structure, function and tissue distribution . This review compares and contrasts these differences, and incorporates into the existing body of knowledge these new breakthroughs.

J Exp Med, 1993 Sep 1, 178(3), 917 - 24
Prokaryotic peptides that block leukocyte adherence to selectins; Rozdzinski E et al.; Pertussis toxin binds target cells through the carbohydrate recognition properties of two subunits, S2 and S3, which share amino acid sequence similarity with the lectin domains of the eukaryotic selectin family . Selectins appear on inflamed endothelial cells and promote rolling of leukocytes by reversibly binding carbohydrates . S2, S3, and synthetic peptides representing their carbohydrate recognition domains competitively inhibited adherence of neutrophils to selectin-coated surfaces and to endothelial cells in vitro . These proteins and peptides also rapidly upregulated the function of the leukocyte integrin CD11b/CD18 . These findings implicate mimicry of eukaryotic selectins by prokaryotic adhesive ligands and link the mechanisms underlying leukocyte trafficking to microbial pathogenesis.

Curr Opin Nephrol Hypertens, 1993 Sep, 2(5), 735 - 43
Structural and functional aspects of P-glycoproteins and related transport proteins; Lepage P et al.; The emergence of multidrug resistance in tumor cells is caused by the expression of P-glycoprotein . P-glycoprotein has a unique structure formed by two homologous halves, each encoding six putative transmembrane segments and one nucleotide binding fold . This structural arrangement has been conserved in a large number of eukaryotic and prokaryotic membrane transport systems, which together form the ATP binding cassette superfamily . These membrane transporters act on different groups of substrates in different cell types and organisms . The combined molecular analysis of these proteins has shed light on the mechanism by which P-glycoprotein acts on structurally unrelated groups of chemotherapeutic drugs and has allowed the identification of the protein domain responsible for the common mechanism of transport and recognition of substrate molecules . The function of P-glycoprotein in normal tissues remains intriguing and is discussed in this review.

Biochemistry, 1993 Aug 24, 32(33), 8582 - 8
Spectrum of DNA--platinum adduct recognition by prokaryotic and eukaryotic DNA-dependent RNA polymerases; Corda Y et al.; Double-stranded DNA oligomers were constructed to evaluate the effect of bifunctional and monofunctional platinum(II) complexes at the level of DNA transcription . They contained a single lesion, which is either a cis-{Pt(NH3)2(d(GpTpG))} intrastrand cross-link, a trans-{Pt(NH3)2(d(GpTpG))} intrastrand cross-link, a cis-{Pt(NH3)2(d(GpC/GpC))} interstrand cross-link, or a (diethylenetriamine)-platinum(II)-dG adduct . The synthetic duplexes were multimerized and then used as templates in dinucleotide-primed reactions catalyzed by prokaryotic or eukaryotic RNA polymerases . Reactions were conducted in the presence of a single triphosphate substrate (single-step addition reaction) or of a combination of triphosphate substrates, permitting elongation of the trinucleotide products to longer RNA chains (productive elongation reaction), respectively . In transcription of the platinated strands, none of the DNA adducts provided an absolute block to formation of a single phosphodiester bond by either Escherichia coli RNA polymerase or wheat germ RNA polymerase II . However, the single-step addition reactions were much more impeded from transcription of bifunctional adduct-containing templates as compared to those containing monofunctional lesions . Productive elongation was irreversibly blocked in transcription of the platinated strand of templates containing a cis-d(G*pTpG*) intrastrand cross-link or a cis-d(G*pC/G*pC) interstrand cross-link . In both cases transcription stopped at the level of the lesion . Termination occurred also several nucleotides before the elongation complexes reached the interstrand cross-link . A substantial amount of the RNA polymerase molecules was able of bypassing the trans-d(G*pTpG*) cross-links . In all the cases single-step addition reactions were enhanced on the template strand complementary to that containing the intrastrand cross-links.(ABSTRACT TRUNCATED AT 250 WORDS)

FEBS Lett, 1993 Aug 23, 329(1-2), 153 - 8
Cloning of a human liver UDP-glucose pyrophosphorylase cDNA by complementation of the bacterial galU mutation; Peng HL et al.; A human liver cDNA clone which encodes the UDP-glucose pyrophosphorylase was isolated by complementation of a bacterial galU mutant . The deduced amino acid sequence of the human enzyme comprised 508 amino acids with a calculated molecular mass of 56,950 . The human enzyme significantly resembles those of potato tuber and slime mold with a homology of 46.6% and 43.2%, respectively, in amino acid sequence . No homology was found between the eukaryotic and the prokaryotic enzymes . Northern blotting analysis revealed that the gene was expressed at the highest level in skeletal muscle, followed by liver, heart and kidney.

J Mol Biol, 1993 Aug 20, 232(4), 1221 - 6
An avian alpha B-crystallin . Non-lens expression and sequence similarities with both small (HSP27) and large (HSP70) heat shock proteins; Lee DC et al.; alpha B-crystallin is multifunctional, serving as both a major structural protein in the lens and a small heat-shock protein (shsp) in other tissues in mammals . Cloning and Northern analysis show similarly that alpha B-crystallin mRNA is present in all mature tissues examined in a bird (Anas platyrhynchos), although there are some differences in the pattern of transcripts seen . Interestingly, sequence analysis not only shows that duck alpha B-crystallin is a member of the shsp family, as expected, but that this family shares more distant similarity with another heat shock protein family, the highly conserved HSP70s of both eukaryotes and prokaryotes . This raises the possibility that large and small hsps may share structural and perhaps functional features.

Gene, 1993 Aug 16, 130(1), 141 - 4
Cloning and sequencing of the dnaK region of Streptomyces coelicolor A3(2); Bucca G et al.; The dnaK homologue of Streptomyces coelicolor A3(2) strain M145 has been cloned and sequenced . Nucleotide sequence analysis of a 2.5-kb region revealed an open reading frame (ORF) encoding a predicted DnaK protein of 618 amino acids (M(r) = 66,274) . The dnaK coding sequence displays extreme codon bias and shows a strong preference for CGY and GGY, for Arg and Gly codons, respectively . The predicted DnaK sequence has a high Lys:Arg ratio which is not typical of streptomycete proteins . The region immediately downstream from dnaK contains an ORF for a GrpE-like protein; the predicted start codon of grpE overlaps the last two codons of dnaK, indicating that the two genes are translationally coupled . This organisation differs from that reported for other prokaryotes.

Eur J Biochem, 1993 Aug 15, 216(1), 177 - 81
Effect of ionizing radiation and topoisomerase II inhibitors on DNA synthesis in mammalian cells; Lallev A et al.; We have used a novel, quantitative approach to study the effect of gamma-radiation and topoisomerase-II inhibitors on the initiation of DNA synthesis in eukaryotic cells . We found out that mild gamma-irradiation caused an almost immediate decrease in the rate of initiation of genomic DNA replication and stimulated DNA repair . This held true for two different cell lines . Ehrlich ascites tumor cells and Friend transformed erythroid cells, although the effect of gamma-radiation on Friend cells was more pronounced . At the same time, the synthesis of mitochondrial DNA was not affected by the irradiation . The effect of topoisomerase-II inhibitors on DNA initiation closely paralleled that of gamma irradiation, but did not stimulate repair . The fact that gamma-radiation and topoisomerase-II inhibitors, two types of agents that differ so profoundly, have practically the same effect on DNA synthesis speaks strongly in favour of the idea that eukaryotic cells have a general mechanism for coping with any disturbances in DNA integrity and chromatin structure . This mechanism is probably similar to the SOS-mechanism of prokaryotic cells and includes, as an early step, a slowdown of the initiation of replicative DNA synthesis.

Anal Biochem, 1993 Aug 15, 213(1), 40 - 8
Recombinant technology as an alternative to chemical peptide synthesis: expression and characterization of HIV-1 Rev recombinant peptides; Lepage P et al.; Recombinant DNA technology has been widely used for the production of proteins in recent years . In this paper, we describe the expression and the purification of two specific peptides corresponding to parts of the human immunodeficiency virus Rev protein . The strategy of this method relies on the chemical synthesis of a pair of two complementary oligodeoxynucleotides corresponding to the coding region of the peptide of interest and the subsequent cloning into a prokaryotic expression vector . Transformation of Escherichia coli with these synthetic gene constructs yielded high production levels of recombinant protein in the bacteria . The recombinant protein was composed of two moieties, one corresponding to an "affinity handle" and the second corresponding to the peptide . Chemical cleavage of the fused protein followed by a combination of affinity chromatography and rp-HPLC led to rapid and convenient peptide purification . Peptide fused to the affinity handle as well as cleaved peptide were fully characterized by N-terminal microsequencing and mass spectrometry . The data presented demonstrate that although the major recombinant products had the expected amino acid composition, we detected unexpected processing such as alternative cleavage within the signal peptide, modified cysteines, and deamidations . These results emphasize the importance of the complete characterization of recombinant products by efficient analytical tools such as N-terminal microsequencing and mass spectrometry.

FEMS Microbiol Lett, 1993 Aug 1, 111(2-3), 147 - 52
Characterisation of a highly repeated DNA sequence from Mycobacterium bovis; Doran TJ et al.; We report characterisation of a novel repeat sequence from a Mycobacterium bovis genomic library . The highly repeated sequence belongs to a family consisting of a 24 base pair (bp) direct repeat (DR), that appears to be organized into clusters on the chromosome . We classify the 24-bp DR into the group of prokaryotic DNA repeats known as the interspersed repetitive sequence elements . The 24-bp DR will be of potential use as a DNA fingerprinting tool in epidemiological studies of M . bovis.

Nat Genet, 1993 Aug, 4(4), 367 - 72
Genomic scanning for expressed sequences in Xp21 identifies the glycerol kinase gene; Guo W et al.; Rapid genomic scanning methods are required to identify expressed sequences and we report an efficient, sensitive and specific approach which relies upon hybridization of an amplified, labeled cDNA library to digested cosmid DNA . We identified expressed sequences within a cosmid in the glycerol kinase (GK) "critical region" of Xp21 that had impressive similarity to prokaryotic GKs . We used this genomic sequence information to clone the human hepatic GK cDNA . Independent confirmation of the identity of this gene was obtained by functional complementation of GK deficient E . coli mutants with a construct containing the complete human X-linked GK coding sequence.

Protein Sci, 1993 Aug, 2(8), 1255 - 62
Cloning, characterization, and expression of the beta subunit of pig heart succinyl-CoA synthetase; Bailey DL et al.; The form of succinyl-CoA synthetase found in mammalian mitochondria is known to be an alpha beta dimer . Both GTP- and ATP-specific isozymes are present in various tissues . We have isolated essentially identical complementary DNA clones encoding the beta subunit of pig heart succinyl-CoA synthetase from both newborn and adult tissues . These cDNAs include a 1.4-kb sequence encoding the cytoplasmic precursor to the beta subunit comprised of 417 amino acid residues including a 22-residue mitochondrial targeting sequence . The cDNA encoding the 395-amino acid, 42,502-Da mature protein was confirmed to be the succinyl-CoA synthetase beta subunit by agreement with the N-terminal protein sequence and by high homology to prokaryotic forms of the beta subunit that were previously cloned (about 45% identical to beta from Escherichia coli) . In contrast to a previous report (Nishimura, J.S., Ybarra, J., Mitchell, T., & Horowitz, P.M., 1988, Biochem . J . 250, 429-434), we found no tryptophan residue to be encoded in the sequence for the mature beta subunit, and this finding is corroborated by the fact that highly purified pig heart succinyl-CoA synthetase shows no tryptophan fluorescence or tryptophan content in amino acid compositional analysis . The cDNA clones encoding the mature pig heart beta subunit and its counterpart alpha subunit were coexpressed in a deletion mutant strain of E . coli . Recovery of succinyl-CoA synthetase activity demonstrated that this combination of subunits forms a productive enzymatic complex having GTP specificity.

Biokhimiia, 1993 Aug, 58(8), 1154 - 75
{DNA-binding lipids: composition and possible functions}; Struchkov VA et al.; The experimental data concerning the composition of DNA-bound lipids of different eukaryotic and prokaryotic cells have been summarized . Using X-ray diffraction patterns, circular dichroism, microcalorimetry, electron microscopy, viscoelastometry and sedimentation methods, it has been proved that the lipids are important integral components of chromosomal DNA . It was shown that the DNA-bound lipids have a specific composition which differs from that of chromatin, nuclear membrane and matrix lipids . The composition of these lipids changes depending on the activity of the genome and the phase of the cell cycle as well as when DNA passes from a supercoiled into a relaxed state . The DNA of cancer cells has a specific composition of the lipid component . The lipids take part in the regulation of transcription . The DNA-bound lipids are hypersensitive target sites for ionizing radiation and anticancer agents . The role of this lipid class in structure-functional organization of chromosomal DNA is discussed.

J Bacteriol, 1993 Aug, 175(16), 4957 - 61
Evidence that the catalytic activity of prokaryote leader peptidase depends upon the operation of a serine-lysine catalytic dyad; Black MT; Leader peptidase (LP) is the enzyme responsible for proteolytic cleavage of the amino acid leader sequence from bacterial preproteins . Recent data indicate that LP may be an unusual serine proteinase which operates without involvement of a histidine residue (M . T . Black, J . G . R . Munn, and A . E . Allsop, Biochem . J . 282:539-543, 1992; M . Sung and R . E . Dalbey, J . Biol . Chem . 267:13154-13159, 1992) and that, therefore, one or more alternative residues must perform the function of a catalytic base . With the aid of sequence alignments, site-specific mutagenesis of the gene encoding LP (lepB) from Escherichia coli has been employed to investigate the mechanism of action of the enzyme . Various mutant forms of plasmid-borne LP were tested for their abilities to complement the temperature-sensitive activity of LP in E . coli IT41 . Data are presented which indicate that the only conserved amino acid residue possessing a side chain with the potential to ionize, and therefore with the potential to transfer protons, which cannot be substituted with a neutral side chain is lysine at position 145 . The data suggest that the catalytic activity of LP is dependent on the operation of a serine-lysine catalytic dyad.

J Immunol, 1993 Aug 1, 151(3), 1214 - 23
Close correlation between Daudi and mycobacterial antigen recognition by human gamma delta T cells and expression of V9JPC1 gamma/V2DJC delta-encoded T cell receptors; Davodeau F et al.; Recent studies have demonstrated that a large fraction of human gamma delta PBL recognize Ag of prokaryotic and eukaryotic origins, respectively found in hydrosoluble mycobacterial extracts and on the Daudi Burkitt's lymphoma cells . The structural basis of the recognition of these Ag have been presently studied in detail, through analysis of a large panel of thymus- and peripheral blood-derived gamma delta T-cell clones . Our results suggest that Daudi and mycobacteria-reactive gamma delta subsets are strictly overlapping and hence that gamma delta T-cell responses against these two Ag are closely related . Daudi cells and mycobacteria were recognized by V gamma 9+V delta 2+, but not by V gamma 9+V delta 2-, V gamma 9-V delta 2+, or V gamma 9-V delta 2- PBL clones . However, not all V gamma 9+V delta 2+ clones were reactive and, in particular: 1) the proportion of Ag-reactive lymphocytes was much lower among thymus- than PBL-derived clones (respectively 24/36 vs 72/73); 2) none of the V gamma 9+V delta 2+ clones expressing a V9J2C2 gamma chain (n = 4) were reactive to Daudi or mycobacteria, indicating that expression of a disulfide-linked TCR is probably a prerequisite for recognition of these Ag; and 3) among V gamma 9+V delta 2+ clones bearing disulfide-linked TCR, almost all (50/53) clones expressing a V9JPC1 gamma chain were reactive, whereas a large fraction (6/10) of those expressing a V9J1C1 gamma chain were weakly or nonreactive . Together, these observations suggest that germline residues specific to V gamma 9, V delta 2, and J gamma P elements directly contribute to recognition of Daudi and mycobacterial Ag . Furthermore, these findings may provide an explanation for coordinate use of these gene elements by a large fraction of gamma delta PBL, through peripheral selection events mediated by ligands identical or structurally related to the above Ag.

Genetics, 1993 Aug, 134(4), 1039 - 44
Antimutator mutations in the alpha subunit of Escherichia coli DNA polymerase III: identification of the responsible mutations and alignment with other DNA polymerases; Fijalkowska IJ et al.; The dnaE gene of Escherichia coli encodes the DNA polymerase (alpha subunit) of the main replicative enzyme, DNA polymerase III holoenzyme . We have previously identified this gene as the site of a series of seven antimutator mutations that specifically decrease the level of DNA replication errors . Here we report the nucleotide sequence changes in each of the different antimutator dnaE alleles . For each a single, but different, amino acid substitution was found among the 1,160 amino acids of the protein . The observed substitutions are generally nonconservative . All affected residues are located in the central one-third of the protein . Some insight into the function of the regions of polymerase III containing the affected residues was obtained by amino acid alignment with other DNA polymerases . We followed the principles developed in 1990 by M . Delarue et al . who have identified in DNA polymerases from a large number of prokaryotic and eukaryotic sources three highly conserved sequence motifs, which are suggested to contain components of the polymerase active site . We succeeded in finding these three conserved motifs in polymerase III as well . However, none of the amino acid substitutions responsible for the antimutator phenotype occurred at these sites . This and other observations suggest that the effect of these mutations may be exerted indirectly through effects on polymerase conformation and/or DNA/polymerase interactions.

Genes Dev, 1993 Aug, 7(8), 1521 - 34
The nonspecific DNA-binding and -bending proteins HMG1 and HMG2 promote the assembly of complex nucleoprotein structures; Paull TT et al.; The mammalian high mobility group proteins HMG1 and HMG2 are abundant, chromatin-associated proteins whose cellular function is not known . In this study we show that these proteins can substitute for the prokaryotic DNA-bending protein HU in promoting the assembly of the Hin invertasome, an intermediate structure in Hin-mediated site-specific DNA inversion . Formation of this complex requires the assembly of the Hin recombinase, the Fis protein, and three cis-acting DNA sites, necessitating the looping of intervening DNA segments . Invertasome assembly is strongly stimulated by HU or HMG proteins when one of these segments is shorter than 104 bp . By use of ligase-mediated circularization assays, we demonstrate that HMG1 and HMG2 can bend DNA extremely efficiently, forming circles as small as 66 bp, and even 59-bp circles at high HMG protein concentrations . In both invertasome assembly and circularization assays, substrates active in the presence of HMG1 contain one less helical turn of DNA compared with substrates active in the presence of HU protein . Analysis of different domains of HMG1 generated by partial proteolytic digestion indicate that DNA-binding domain B is sufficient for both bending and invertasome assembly . We suggest that an important biological function of HMG1 and HMG2 is to facilitate cooperative interactions between cis-acting proteins by promoting DNA flexibility . A general role for HMG1 and HMG2 in chromatin structure is also suggested by their ability to wrap DNA duplexes into highly compact forms.

Mol Cell Biol, 1993 Aug, 13(8), 4784 - 92
Coexpression of two closely linked avian genes for purine nucleotide synthesis from a bidirectional promoter; Gavalas A et al.; Two avian genes encoding essential steps in the purine nucleotide biosynthetic pathway are transcribed divergently from a bidirectional promoter element . The bidirectional promoter, embedded in a CpG island, directs coexpression of GPAT and AIRC genes from distinct transcriptional start sites 229 bp apart . The bidirectional promoter can be divided in half, with each half retaining partial activity towards the cognate gene . GPAT and AIRC genes encode the enzymes that catalyze step 1 and steps 6 plus 7, respectively, in the de novo purine biosynthetic pathway . This is the first report of genes coding for structurally unrelated enzymes of the same pathway that are tightly linked and transcribed divergently from a bidirectional promoter . This arrangement has the potential to provide for regulated coexpression comparable to that in a prokaryotic operon.

Protein Eng, 1993 Aug, 6(6), 621 - 8
Repeat of a helix-turn-helix module in DNA-binding proteins; Yura K et al.; Helix-turn-helix motif is one of the common motifs observed in DNA-binding proteins . The motif interacts with DNA double helix and recognizes specific base sequences . It is assumed that the helix-turn-helix motif appears only once in seven prokaryotic transcriptional repressors of which 3-D structures have been determined by X-ray crystallographic studies . These prokaryotic repressors consist of several alpha-helices connected with turns . We report here that these repressors are decomposable into helix-turn-helix modules and their connectors . A module is defined as a compact structural unit with consecutive amino acid residues in a globular protein . Each of the helix-turn-helix motifs in the seven proteins corresponds approximately to a single helix-turn-helix module consisting of approximately 13 amino acids . Identification of modules of seven prokaryotic repressors and comparisons of their tertiary structures led to the conclusion that three of these DNA-binding proteins contain more than one helix-turn-helix module with a structure similar to the helix-turn-helix motif . The difference in module organization of these DNA-binding proteins paves the way for further classification of the DNA-binding proteins with the helix-turn-helix motif . The structural repertoire of these transcriptional regulators was increased through different utilizations in the number of helix-turn-helix and other modules . The difference in DNA base recognition ability in these helix-turn-helix modules is ascribed to a difference in size of a side chain at the fifth residue from Gly, on the turn.

Mol Microbiol, 1993 Aug, 9(4), 787 - 801
SyrD is required for syringomycin production by Pseudomonas syringae pathovar syringae and is related to a family of ATP-binding secretion proteins; Quigley NB et al.; The syrD gene of Pseudomonas syringae pathovar syringae strain B301D-R was characterized and sequenced . The syrD open reading frame is 1695 bp long and encodes a predicted protein, SyrD, of approximately 63 kDa . Database searches revealed that SyrD shares a high degree of similarity with the ATP-binding cassette (ABC) superfamily of transporter proteins which are responsible for specific nutrient uptake and for secretion of certain cellular products in prokaryotes, and for multiple drug resistance in mammals . The amino acid sequence homology between SyrD and the ABC proteins was greatest at the conserved residues which constitute the ATP-binding cassette of these proteins; these residues lie in the hydrophilic C-terminal half of SyrD . The N-terminus of SyrD is predicted to be hydrophobic and to contain six membrane-spanning alpha-helices . syrD mutants of strain B301D-R were significantly less virulent than other syr mutants, were deficient in four large polypeptides thought to be components of a syringomycin synthetase complex, and showed reduced expression of a syrB-lacZ reporter gene fusion in trans . It is proposed that SyrD is a cytoplasmic membrane protein that functions as an ATP-driven efflux pump for the secretion of syringomycin.

Curr Opin Immunol, 1993 Aug, 5(4), 492 - 6
Cellular immunity to intracellular bacteria; Pamer EG; Great progress has been made in understanding the mechanisms bacteria use to invade, survive and move within eukaryotic cells . It is clear that bacteria have found ways to manipulate host cell signal transduction pathways and the cytoskeleton to their advantage . To defend against prokaryotic invaders, the immune system has evolved mechanisms for the specific recognition of bacterial antigens.

Biochem J, 1993 Aug 1, 293 ( Pt 3), 829 - 33
Common sequence motifs coding for higher-plant and prokaryotic O-acetylserine (thiol)-lyases: bacterial origin of a chloroplast transit peptide?
Rolland N, Job D, Douce R.
A comparison of the amino acid sequence of O-acetylserine (thiol)-lyase (EC 4.2.99.8) from Escherichia coli and the isoforms of this enzyme found in the cytosolic and chloroplastic compartments of spinach (Spinacia oleracea) leaf cells allows the essential lysine residue involved in the binding of the pyridoxal 5'-phosphate cofactor to be identified . The results of further sequence comparison of cDNAs coding for these proteins are discussed in the frame of the endosymbiotic theory of chloroplast evolution . The results are compatible with a mechanism in which the chloroplast enzyme originated from the cytosolic enzyme and both plant genes originated from a common prokaryotic ancestor . The comparison also suggests that the 5'-non-coding sequence of the bacterial gene was transferred to the plant cell nucleus and that it has been used to create the N-terminal portions of both plant enzymes, and possibly the transit peptide of the chloroplast enzyme.

Biotechnology (N Y), 1993 Aug, 11(8), 933 - 6
Recombinant baculovirus vectors expressing glutathione-S-transferase fusion proteins; Davies AH et al.; Recombinant baculoviruses are a popular means of producing heterologous protein in eukaryotic cells . Purification of recombinant proteins away from the insect cell background can, however, remain an obstacle for many developments . Recently, prokaryotic fusion protein expression systems have been developed allowing single-step purification of the heterologous protein and specific proteolytic cleavage of the affinity tag moiety from the desired antigen . Here we report the introduction of these attributes to the baculovirus system . "Baculo-GEX" vectors enable baculovirus production of fusion proteins with the above advantages, but in a eukaryotic post-translational processing environment . Glutathione-S-transferase (GST) fusions are stable cytoplasmic proteins in insect cells and may therefore be released by sonication alone, avoiding the solubility problems and detergent requirements of bacterial systems . Thus large amounts of authentic antigen may be purified in a single, non-denaturing step.

Cell Immunol, 1993 Aug, 150(1), 90 - 100
Novel regulation of an MHC class I gene response to interferon-gamma; Schwarz DA et al.; The ability of IFN-gamma to increase the expression of MHC class I gene products is likely to enhance cytolytic T lymphocyte recognition of viral pathogens and tumor cells . The murine lymphoma AKR SL3-cl.F AZR (SL3-cl.F) responds aberrantly to treatment with interferon-gamma such that H-2Dk surface expression is augmented, but H-2Kk expression remains at constitutive levels . Somatic cell fusions have been used to demonstrate that the lesion responsible for this phenotype is cis-dominant, implicating a primary lesion within the SL3-cl.F H-2Kk gene . In this communication, we have used PCR to analyze the nucleotide sequence in regions of the SL3-cl.F H-2Kk promoter known to contain interferon-responsive enhancer elements . Comparison of the SL3-cl.F H-2Kk sequences to known consensus elements revealed complete identity . In order to identify the lesion responsible for the SL3-cl.F phenotype, two H-2Kk genomic clones were independently isolated from SL3-cl.F . Each clone exists as a 10.5-kbp EcoRI fragment containing the entire structural gene . The site of transcription initiation is at the center of this fragment; therefore, all regulatory elements within 5 kbp of the transcript start site which could alter steady-state message levels are included . Interestingly, IFN-gamma-augmented expression of the H-2Kk gene was restored following DNA-mediated transfection of either of these clones into fibroblast cell lines and the parental cell line SL3-cl.F . Because isolation of these clones required passage of the DNA through a prokaryotic host, which alters the pattern of DNA methylation, there was the possibility that demethylation was responsible for the newly acquired IFN-gamma-responsive phenotype . Treatment of SL3-cl.F with 5-azacytidine, which inhibits de novo methylation, did not restore IFN-gamma-augmented expression, however, thus excluding H-2Kk specific methylation as a potential mechanism . Collectively, these data demonstrate that the alteration responsible for the phenotype observed in SL3-cl.F does not involve known transcriptional regulatory elements . Potential mechanisms which might account for the mutant phenotype are discussed.

Mutat Res, 1993 Aug, 294(2), 187 - 98
DNA repair and mutation: recent advances . A report of the meeting of the British Photobiology Society, London, 21 December 1992; Strike P; The 1992 British Photobiology Society DNA Repair meeting was held at the University of East London on December 21st . As is now customary, the meeting was very well supported, and a full program of twenty one talks were presented on topics related to DNA repair and mutation . The usual format of the meeting was maintained, with all talks being voluntary contributions, there being no specially invited speakers . The continued interest in this field of research was reflected by the large number of offered talks, that contributed to a program covering both prokaryotic and eukaryotic systems.

Mutat Res, 1993 Aug, 294(2), 167 - 77
Molecular spectrum of mutations induced at the HPRT locus by a cross-linking agent in human cell lines with different repair capacities; Papadopoulo D et al.; Molecular characterization of mutations photoinduced by a cross-linking agent, 4,5',8-trimethylpsoralen (Me3Pso), in normal human lymphoblasts was conducted in parallel with lymphoblasts derived from Fanconi anemia patients . Such cells have been previously described to be impaired in repair of psoralen photolesions . The endogenous HPRT locus was used as a target gene . The treatment of cells with Me3Pso in combination with 365 nm irradiation leads to the formation of interstrand cross-links, and specific monoadducts . Our analysis revealed that the mutagenic processing of Me3Pso photoadducts in normal human cells results essentially in base substitutions (84%) . These are localized to sequences shown previously to be favored for the formation of Me3Pso monoadducts . The mutagenic processing of the same lesions in Fanconi anemia cells results in fewer base substitutions (22%), with deletions (66%) being the predominant class of mutation . In contrast to prokaryotic systems, frameshifts are poorly represented among Me3Pso induced mutations in human cells . In spite of important differences between the kinds of mutations observed in the two cell lines, our analysis reveals similarities in the type of base substitutions and their sequence distribution . In both normal and Fanconi anemia cell lines mutations, mostly targeted on thymine residues, are preferentially located on the non-transcribed strand.

Biochem Biophys Res Commun, 1993 Jul 30, 194(2), 642 - 6
Hyperosmotic stress induces immediate-early gene expression in ventricular adult cardiomyocytes; Wollnik B et al.; Mammalian cells possessing osmosensors have long been described in brain and kidney . The genetic basis of the response to hyperosmotic stress has been well characterized in prokaryotes . In contrast, the genetic response of eukaryotic cells is poorly understood . Therefore we investigated the effect of hypertonic NaCl and sucrose solutions on the transcriptional activation of the immediate-early genes (IEGs) egr-1 and c-fos in isolated ventricular adult rat cardiomyocytes . We observed that even small increases in osmolarity to 315 +/- 5 mosmol/l and 370 +/- 8 mosmol/l by hypertonic NaCl solution resulted in dose-dependent induction of egr-1 (4-and 5-fold) and c-fos (3-and 4-fold), respectively . Hypertonic sucrose solution had the same effect on egr-1 and c-fos mRNA levels while increased sucrose concentration under isotonic conditions had no effect . Cardiomyocytes exposed to hypertonic media did not significantly shrink as shown by a cell length measurement . We conclude that isolated adult cardiomyocytes possess an osmoreceptor mechanism which is able to sense even slight changes in osmolarity and to translate these into a transcriptional response of the myocardial IEG program.

Proc Natl Acad Sci U S A, 1993 Jul 15, 90(14), 6601 - 5
Effects of abasic sites and DNA single-strand breaks on prokaryotic RNA polymerases; Zhou W et al.; Abasic sites are thought to be the most frequently occurring cellular DNA damage and are generated spontaneously or as the result of chemical or radiation damage to DNA . In contrast to the wealth of information that exists on the effects of abasic sites on DNA polymerases, very little is known about how these lesions interact with RNA polymerases . An in vitro transcription system was used to determine the effects of abasic sites and single-strand breaks on transcriptional elongation . DNA templates were constructed containing single abasic sites or nicks placed at unique locations downstream from two different promoters and were transcribed by SP6 and Escherichia coli RNA polymerases . SP6 RNA polymerase is initially stalled at abasic sites with subsequent, efficient bypass of these lesions . E . coli RNA polymerase also bypassed abasic sites . In contrast, single-strand breaks introduced at abasic sites completely blocked the progression of both RNA polymerases . Sequence analysis of full-length transcripts revealed that SP6 and E . coli RNA polymerases insert primarily, if not exclusively, adenine residues opposite to abasic sites . This finding suggests that abasic sites may be highly mutagenic in vivo at the level of transcription.

J Immunol, 1993 Jul 15, 151(2), 907 - 15
Effect of in vivo inhibition of nitric oxide production in murine leishmaniasis; Evans TG et al.; In vitro experiments suggest that cytokine induced nitric oxide (NO) synthesis from L-arginine is a major effector mechanism against prokaryotic and eukaryotic intracellular pathogens . N omega monomethyl L-arginine (MLA), an active site inhibitor of the cytokine induced NO synthase, inhibits cytokine induced resistance of mammalian cells to intracellular microbes in vitro . In our experiments, we show that Leishmania infection markedly increases NO synthesis in the genetically resistant C3H/HeN mouse strain . In addition, administration of 50 mM MLA in the drinking water inhibits endogenous NO synthesis as well as natural resistance to footpad Leishmania infections in both susceptible BALB/c and resistant C3H/HeN mice . Leishmania parasites continued to proliferate in MLA-treated C3H/HeN mice after footpad inoculation . Similarly C3H/HeN mice treated for 3 wk with MLA had an increased parasite load and sloughing of the footpad . Footpad size of C3H/HeN mice not treated with MLA returned to base-line diameter and the regional nodes contained few amastigotes . These in vivo effects were paralleled by endogenous NO synthesis (high when Leishmania was controlled and low when Leishmania was not controlled) . In addition, inhibition of NO synthesis in Leishmania-infected mice leads to a cachectic state not caused by infection alone or inhibition of NO synthesis alone . The cachexia appeared to be due to decreased food intake that occurs when NO synthesis is inhibited in Leishmania-infected mice . Our results show that cytokine induced NO has a major effector role in resistance of murine hosts to Leishmania infection.

Biochem Mol Biol Int, 1993 Jul, 30(3), 453 - 60
Glycogen-bound protein in lower eukaryote and prokaryote; Goldraij A et al.; The proteoglycogen fraction of Neurospora crassa was purified and subjected to radioiodination with {125I}iodide . Amylolysis of the polysaccharide moiety led to the isolation of a labelled 31 kDa-protein . The NH2-terminal amino acid sequence of 10 residues of the 31 kDa-protein was determined . A 31 kDa-protein was also bound to glycogen in Escherichia coli . Proteoglycogen has not been heretofore found in any primitive unicellular organism.

Mol Plant Microbe Interact, 1993 Jul-Aug, 6(4), 467 - 73
Cloning and targeted gene disruption of XYL1, a beta 1,4-xylanase gene from the maize pathogen Cochliobolus carbonum; Apel PC et al.; The gene, XYL1, encoding the major extracellular endo-beta 1,4-xylanase from the maize pathogen Cochliobolus carbonum was cloned using a synthetic, degenerate oligonucleotide based on a tryptic fragment from the purified enzyme . The deduced product of XYL1 has a M(r) of 20,869 and a predicted pI of 9.1, in good agreement with the measured M(r) and pI of the purified enzyme . The XYL1 product has strong amino acid identity to seven endo-beta 1,4-xylanases from six prokaryotes but no obvious similarity to 10 other prokaryotic endoxylanases or a yeast endoxylanase . An internal fragment of the gene was used to create a specific xylanase mutant by transformation-mediated gene disruption via homologous recombination . Total extracellular xylanase activity in the mutant was reduced by 85-94% . When analyzed by cation exchange HPLC, culture filtrates of the mutant and wild type had identical protein profiles, but the mutant lacked the major peak of UV absorption corresponding to the major xylanase activity . Xylanase II activity was also missing in the mutant, but xylanase III activity was still present . The XYL1 mutant grew as well as the wild type on sucrose, on corn cell walls, and on xylan . The pathogenicity of the mutant was indistinguishable from the wild type, indicating that XYL1 is not required for pathogenicity.

J Periodontol, 1993 Jul, 64(7), 630 - 6
Inhibitory effect of tea catechins on collagenase activity; Makimura M et al.; A major purpose of this study was to examine inhibitory effect of the catechin derivatives from Japanese green tea Camellia sinensis on collagenase activity . The crude tea catechins, which contain (+)-catechin (C), (-)-epicatechin (EC), (+)-gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg), were tested for their ability to inhibit the prokaryotic and eukaryotic cell derived collagenase activities . Among the tea catechins tested, ECg and EGCg showed the most potent inhibitory effect on collagenase activity when an optimal concentration of tea catechins (100 micrograms/ml) was added to reaction mixture containing collagenase and collagen . Preincubation of collagenase with tea catechins reduced the collagenase activity as well . In contrast to ECg and EGCg, the other four tea catechins (C, EC, EGC, and GC) did not show any collagenase inhibitory effect . Our results suggest that the steric structure of 3-galloyl radical is important for the inhibition of collagenase activity . The collagenase activity in the gingival crevicular fluid from highly progressive adult periodontitis was completely inhibited by the addition of tea catechins . These results demonstrated that tea catechins containing galloyl radical possess the ability to inhibit both eukaryotic and prokaryotic cell derived collagenase.

Curr Genet, 1993 Jul-Aug, 24(1-2), 12 - 20
Expression enhancement of the Tn5 neomycin-resistance gene by removal of upstream ATG sequences and its use for probing heterologous upstream activating sequences in yeast; Yagi S et al.; We have constructed a series of promoter or upstream activating sequence (UAS)-probe plasmids carrying the Tn5-derived neomycin resistance gene whose seven additional ATG codons in the 5'-untranslated region were completely or partially removed . When the deleted version of the neo sequence retaining only one additional ATG (NeoD) was expressed under the control of a TDH3 promoter whose UAS was deleted, the transformed cells were unable to grow at a low concentration of the antibiotic G418 . In contrast with this, yeast cells expressing the NeoC sequence and having no additional ATG exhibited a high level of G418-resistance . Moreover, the UAS-probe system using NeoD has been successfully applied for the identification of several E . coli DNA sequences that clearly function as UASs in yeast cells . Two of these prokaryotic sequences with UAS activity were identified as a part of the coding region of the tgt and the hydG gene, respectively.

Mol Gen Genet, 1993 Jul, 240(1), 92 - 102
Overproduction of, and interaction within, bifunctional domains from the amino- and carboxy-termini of the pentafunctional AROM protein of Aspergillus nidulans; Moore JD et al.; The pentafunctional AROM protein in Aspergillus nidulans and other fungi catalyses five consecutive enzymatic steps leading to the production of 5-enolpyruvylshikimate 3-phosphate (EPSP) in the shikimate pathway . The AROM protein has five separate enzymatic domains that have previously been shown to display a range of abilities to fold and function in isolation as monofunctional enzymes . In this communication, we report (1) the stable overproduction of a bifunctional protein containing the 3-dehydroquinate (DHQ) synthase and EPSP synthase activities in Escherichia coli to around 10% of the total cell protein; (2) that both the DHQ synthase and EPSP synthase activities in the overproduced fragment are enzymatically active as judged by their ability to complement aroA and aroB mutants of E . coli; (3) that the EPSP synthase domain is only enzymatically active when covalently attached to the DHQ synthase domain (the cis arrangement) . When DHQ synthase and EPSP synthase are produced concomitantly by transcribing sequences encoding the individual domains from separate plasmids in the same bacterial cell (the trans arrangement) no overproduction or enzyme activity can be detected for the EPSP synthase domain; (4) the EPSP synthase domain can be stably overproduced as a fusion protein with glutathione S-transferase (GST), however the EPSP synthase in this instance is enzymatically inactive; (5) a protein containing an enzymatically inactive DHQ synthase domain in the cis arrangement with EPSP synthase domain is stably overproduced with enzymatically active EPSP synthase; (6) the two C-terminal domains of the AROM protein specifying the 3-dehydroquinase and shikimate dehydrogenase domains can be overproduced in A . nidulans using a specially constructed expression vector . This same bi-domain fragment however is not produced in E . coli when identical coding sequences are transcribed from a prokaryotic expression vector . These data support the view that multifunctional/multidomain proteins do not solely consist of independent units covalently linked together, but rather that certain individual domains interact to varying degrees to stabilise enzyme activity.

J Bacteriol, 1993 Jul, 175(13), 4025 - 35
Anabaena sp . strain PCC 7120 bifA gene encoding a sequence-specific DNA-binding protein cloned by in vivo transcriptional interference selection; Wei TF et al.; VF1 is a DNA-binding protein from the cyanobacterium Anabaena sp . strain PCC 7120 . VF1 was originally identified on the basis of its binding affinity to the upstream region of xisA, which encodes a heterocyst-specific site-specific recombinase . VF1 also binds to the glnA, rbcL, and nifH promoters in vitro, suggesting that VF1 interacts with genes expressed in both vegetative cells and heterocysts . The role of VF1 in regulating gene expression in PCC 7120 is unknown . As a step towards the goal of understanding the role of VF1 in regulating gene expression, we have cloned the bifA gene by using a genetic selection strategy . bifA encodes a protein, BifA, that has chromatographic and DNA-binding properties indistinguishable from those of VF1 . The cloning strategy was based on a transcriptional interference assay in which a strong synthetic promoter, conII, interferes with the expression of an aadA gene, which provides resistance to spectinomycin and streptomycin (S . J . Elledge, P . Sugiono, L . Guarente, and R . W . Davis, Proc . Natl . Acad . Sci . USA 86:3689-3693, 1989) . A selection plasmid, pAM994, which has the conII promoter negatively regulated by a VF1-binding site, was used to enrich for VF1-producing clones from an expression library containing PCC 7120 DNA fragments . Mobility shift assays were used to identify a 672-bp open reading frame that encoded VF1-like binding activity . The deduced BifA amino acid sequence shows 77% identity to NtcA, which is a global regulator involved in nitrogen control in Synechococcus sp . strain PCC 7942 . Both BifA and NtcA belong to the cyclic AMP receptor protein (CRP) family of prokaryotic regulatory proteins . Genes similar to envM, hisB, and ORF60-5 were found near the bifA gene.

Genes Dev, 1993 Jul, 7(7A), 1244 - 53
Characterization of the Caenorhabditis elegans Tc1 transposase in vivo and in vitro; Vos JC et al.; We have investigated the function of the Tc1A gene of the mobile element Tc1 of Caenorhabditis elegans . Tc1 is a member of a family of transposons found in several animal phyla, such as nematodes, insects, and vertebrates . Two lines of evidence show that Tc1A encodes the transposase of Tc1 . First, forced expression of the Tc1A protein in transgenic nematodes results in an enhanced level of transposition of endogenous Tc1 elements . Second, DNase I footprinting and gel retardation assays show that Tc1A binds specifically to the inverted repeats at the ends of the element and that the Tc1A recognition site is located between base pairs 5 and 26 from the ends of Tc1 . Functional dissection of the transposase shows the presence of two distinct DNA-binding domains . A site-specific DNA-binding domain is contained within the amino-terminal 63 residues of Tc1A; this region shows sequence similarity to the prokaryotic IS30 transposase . A second, general DNA-binding domain is located between amino acids 71 and 207 . Our results suggest that Tc1 is more similar to prokaryotic insertion elements than to eukaryotic transposons such as P elements in Drosophila or Ac and En-1 in plants.

Trends Genet, 1993 Jul, 9(7), 246 - 9
Mutagenesis by 8-oxoguanine: an enemy within; Grollman AP et al.; The presence of reactive oxygen species in cells ensures that the oxidatively damaged base 8-oxoguanine will be generated at high frequency in the DNA of all living organisms . DNA damage threatens genomic integrity: enzymes have evolved that protect prokaryotes and eukaryotes from the mutagenic effect of this ubiquitous lesion.

J Photochem Photobiol B, 1993 Jul, 19(2), 87 - 96
Properties and biological functions of the NTH and FPG proteins of Escherichia coli: two DNA glycosylases that repair oxidative damage in DNA; Boiteux S; Oxidative damage to DNA is one of the most important causes of spontaneous mutations and may play a role in aging and related diseases, such as cancer, in humans . Oxidative damage results from the attack of biomolecules by free radicals and reactive oxygen species formed as byproducts of normal cell metabolism or during oxidative stress . To counteract the lethal and mutagenic effects of oxidative lesions in DNA, cells have developed defence strategies including DNA repair systems . In Escherichia coli, the repair of oxidized bases in DNA is mostly mediated by the base excision repair pathway . The first step in this DNA repair pathway is catalysed either by the NTH protein which excises oxidized pyrimidines or by the FPG protein which excises oxidized purines . The nucleotide excision repair pathway mediated by the UvrABC complex may also play a role when the DNA glycosylases are inactive or saturated . This review summarizes the structural and catalytic properties of the NTH and FPG proteins of Escherichia coli and presents evidence to indicate that these two enzymes constitute an important component of the cellular defence against oxidative stress in prokaryotes and eukaryotes.

Chem Res Toxicol, 1993 Jul-Aug, 6(4), 425 - 9
Expression in Escherichia coli of the flavin-containing monooxygenase D (form II) from adult human liver: determination of a distinct tertiary amine substrate specificity; Lomri N et al.; The cDNA for a major component of the family of flavin-containing monooxygenases (FMOs) present in adult human liver (i.e., HLFMO-D) has been cloned and expressed in a prokaryotic system . Escherichia coli strain NM522 was transformed with pTrcHLFMO-D, and the HLFMO-D cDNA was expressed under the control of the Trc promoter . A variety of tertiary amine substrates {i.e., chlorpromazine and 10-{(N,N-dimethylamino)alkyl}- 2-(trifluoromethyl)phenothiazines} were efficiently oxygenated by HLFMO-D cDNA expressed in E . coli or by adult human liver microsomes . Approximate dimensions of the substrate binding channel for both adult human liver microsomal FMO and cDNA-expressed HLFMO-D were apparent from an examination of the N-oxygenation of a series of 10-{(N,N-dimethylamino)alkyl}-2-(trifluoromethyl)phenothiazines . The substrate regioselectivity studies suggest that adult human liver FMO form D possesses a distinct substrate specificity compared with form A FMO from animal hepatic sources . It is likely that the substrate specificity observed for cDNA-expressed adult human liver FMO-D may have consequences for the metabolism and distribution of tertiary amines and phosphorus- and sulfur-containing drugs in humans and may provide insight into the physiologic substrate(s) for adult human liver FMO.

Can J Microbiol, 1993 Jul, 39(7), 718 - 22
Opsonin-independent adherence and intracellular development of Legionella pneumophila within U-937 cells; Rodgers FG et al.; Legionella pneumophila adhered to and multiplied intracellularly in the human histiocytic lymphoma U-937 cell line . The infectious process was evaluated by viable bacterial cell colony counts and documented by transmission and scanning electron microscopy . In the absence of opsonins, wash-resistant bacterial adherence to host cells occurred within 1 h and attachment of 1 or 2 organisms per U-937 host cell involved close surface interactions at the prokaryotic and eukaryotic membranes . Intracellular multiplication of bacteria was maximal by 24 h after inoculation of cell monolayers . Release of L . pneumophila from these cells appeared as a lytic process that resulted in an increase in the numbers of microorganisms in the extracellular fluids and a concomitant decline in the number of intracellular bacteria . The course of cellular infection was completed by 72 h . The cellular and ultrastructural events of L . pneumophila adherence and uptake by U-937 cells in the absence of antibody or complement have been defined . In addition, this work further establishes the U-937 cell as a suitable model for investigating Legionella--host cell interactions.

J Mol Evol, 1993 Jul, 37(1), 71 - 6
Molecular evolutionary analysis based on the amino acid sequence of catalase; von Ossowski I et al.; Heme-containing catalase sequences from 20 different organisms representing prokaryotes, fungi, animals, and plants have been compiled for phylogenetic reconstruction . Phylogenies based on distance and parsimony analysis show that fungal and animal catalases can be derived from one ancestor, whereas bacterial catalases fail to form a monophyletic group . Plant catalases appear to form a second class of catalases that arose independently from a possible prokaryotic ancestor.

Genetics, 1993 Jul, 134(3), 847 - 58
Codon usage bias and base composition of nuclear genes in Drosophila; Moriyama EN et al.; The nuclear genes of Drosophila evolve at various rates . This variation seems to correlate with codon-usage bias . In order to elucidate the determining factors of the various evolutionary rates and codon-usage bias in the Drosophila nuclear genome, we compared patterns of codon-usage bias with base compositions of exons and introns . Our results clearly show the existence of selective constraints at the translational level for synonymous (silent) sites and, on the other hand, the neutrality or near neutrality of long stretches of nucleotide sequence within noncoding regions . These features were found for comparisons among nuclear genes in a particular species (Drosophila melanogaster, Drosophila pseudoobscura and Drosophila virilis) as well as in a particular gene (alcohol dehydrogenase) among different species in the genus Drosophila . The patterns of evolution of synonymous sites in Drosophila are more similar to those in the prokaryotes than they are to those in mammals . If a difference in the level of expression of each gene is a main reason for the difference in the degree of selective constraint, the evolution of synonymous sites of Drosophila genes would be sensitive to the level of expression among genes and would change as the level of expression becomes altered in different species . Our analysis verifies these predictions and also identifies additional selective constraints at the translational level in Drosophila.

J Clin Endocrinol Metab, 1993 Jul, 77(1), 134 - 8
A 12-kilodalton N-glycosylated growth hormone-related peptide is present in human pituitary extracts; Diaz MJ et al.; This study was designed to investigate whether N-linked glycosylation could account for the presence of glycosylated GH forms (G-GH) in human pituitary extracts . The study was carried out in commercially available pituitary GH preparations (pitGH) . Recombinant GHs obtained from eu- or prokaryotic cells were used as controls . Radioiodinated GHs were incubated in tubes containing Concanavalin-A (Con-A) attached to a Sepharose 4B matrix . Pituitary G-GH forms were selectively displaced from Con-A by adding N-acetyl-D-glucosamine or methyl-alpha-D-mannopyranoside and electrophoresed . Autoradiographies of these gels identified a 12-kilodalton (12K) band as the glycosylated form . Due to the fact that this peptide was partially immunoprecipitated with an anti-GH serum and the absence of detectable PRL in the pitGH extracts, it would indicate that such a glycopeptide was GH related . Endoglycosidase-F treatment of pitGH extracts induced a decrease in the mol wt of that 12K peptide, as indicated by the changes observed in its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis . This demonstrated that the sugar moieties were N-linked . When recombinant GHs were assayed by a similar method, no specific binding to Con-A was detected . NH2-terminal amino acid sequence analysis from the 12K band demonstrated that this band was composed by three peptides . Peptide 1 corresponds to the GH-N 102-119 sequence . Interestingly, peptide 2 exhibits GH-V 1-18 sequence, while peptide 3 seems to be a novel GH-related peptide . Taken together, these data suggest that the pituitary G-GH form found in human pituitary extracts is derived not from the "normal" GH-N gene, but, rather, from the GH-V gene or another unidentified gene . The potentially important pathophysiological implications of this finding need to be investigated.

Plant Physiol, 1993 Jul, 102(3), 843 - 50
Characteristics of an Hsp70 homolog localized in higher plant chloroplasts that is similar to DnaK, the Hsp70 of prokaryotes; Wang H et al.; Members of the 70-kD heat-shock protein (Hsp70) family are important cellular factors that are thought to mediate protein folding and assembly . A chloroplast-localized Hsp70 homolog (Chsp70) was recently identified based on its similarity to DnaK, the Hsp70 homolog of Escherichia coli (D . Amir-Shapira, T . Leustek, B . Dalie, H . Weissbach, N . Brot {1990} Proc Natl Acad Sci USA 87: 1749-1752) . To learn more about the function of Chsp70, we purified the protein from Spinacia oleracea chloroplasts by ATP-agarose affinity chromatography . A single, 75,000-D protein was isolated which becomes phosphorylated on a threonine residue when incubated with {gamma-32P}ATP and 10 mM Ca2+, a property similar to DnaK . Chloroplast fractionation and immunoblot analysis showed that Chsp70 is a soluble stromal protein . Chsp70-specific antiserum was used to clone a partial cDNA that shows greater homology with Hsp70 from prokaryotes than with cytoplasmic Hsp70 from eukaryotes . The antiserum and cDNA were used to study Chsp70 expression . Following heat shock of spinach seedlings at 37 degrees C, Chsp70 synthesis increase 12-fold, the level of Chsp70 mRNA increases 5-fold, and the level of Chsp70 protein increases less than 2-fold . Chsp70 is constitutively expressed in all spinach tissues, indicating that it is likely to be localized in all plastid types . The highest levels occur in seeds, leaves, florets, and seedlings grown in the light . Lower levels occur in roots, stems, and etiolated seedlings.

Z Naturforsch {C}, 1993 Jul-Aug, 48(7-8), 595 - 602
Survey of the taxonomic and tissue distribution of microsomal binding sites for the non-host selective fungal phytotoxin, fusicoccin; Meyer C et al.; The recent identification of the fusicoccin-binding protein (FCBP) in plasma membranes from monocotyledonous and dicotyledonous angiosperms has opened the basis for an elucidation of the toxin's mechanism(s) of action and indicated a widespread occurrence of the FCBP in plants . Results of a detailed taxonomic survey of fusicoccin-binding sites are reported . Binding sites were not found in prokaryotes, animal tissues, fungi and algae including the most direct extant ancestors of the land plants (Coleochaete) . From the Psilotales (Psilophytatae) to the monocotyledonous angiosperms, all taxa analyzed possessed high-affinity microsomal fusicoccin-binding sites . A heterogeneous picture emerged for the Bryophyta . Anthoceros crispulus (Anthocerotae), the only hornwort available to study, lacked fusicoccin binding . Within the Hepaticae as well as the Musci, species lacking and species exhibiting toxin binding were found . The binding site thus seems to have emerged very early in the evolution of the land plants . The tissue distribution of fusicoccin-binding sites was studied in Vicia faba L . shoots . All tissues analyzed showed fusicoccin binding, although not to the same extent . On a per-cell basis, guard cells were found to contain, compared to mesophyll cells, a nine-fold higher number of binding sites . Based on cell surface area, the site density is by a factor of 32 higher in guard cells than in mesophyll cells . Tissue specific expression of the binding sites is suggested by these findings.

Biochem Cell Biol, 1993 Jul-Aug, 71(7-8), 401 - 5
Effect of chicken lysozyme signal peptide alterations on secretion of human lysozyme in Saccharomyces cerevisiae; Tsuchiya Y et al.; To investigate the structural requirements and functions of the N-terminal and the C-terminal regions of the chicken lysozyme signal peptide, the amino acids of each region were altered . The replacement of Gly(-1) and Leu(-2) with Pro(-1) and Ala(-2) or Val(-2), respectively, resulted in the complete shift of the cleavage site from position -1 to -2 in yeast (Saccharomyces cerevisiae) . This shows that the introduction of a turn-promoting residue like Pro makes it possible to control the cleavage site of the signal peptide . Deletion of the positive charge and introduction of a negative charge in the N-terminal region decreased the lytic activity of secreted human lysozyme (HLY) and processing efficiency of preHLY, but the length and additional positive charge in this region had little influence . This suggests that the length of the N-terminal region scarcely influences the function of the signal peptide and that this region possibly interacts with the endoplasmic reticulum membrane to initiate the translocation of preprotein, similar to prokaryotic signal peptide . However, it needs only minimum positive charge for its function.

EMBO J, 1993 Jul, 12(7), 2901 - 12
One member of a gro-ESL-like chaperonin multigene family in Bradyrhizobium japonicum is co-regulated with symbiotic nitrogen fixation genes; Fischer HM et al.; This report is concerned with the structural characterization and genetic regulation of new bacterial groES and groEL chaperonin genes, and presents two novelties . The first is the discovery that the nitrogen fixing soybean root nodule bacterium, Bradyrhizobium japonicum, unlike all other prokaryotes investigated so far, possesses a multigene family consisting of five very similar, though not identical, groESL-like genes . The second novelty relates to the finding that these five homologues are expressed to different degrees and, in particular, that one family member (namely groESL3) is induced by a mechanism that does not involve the well-known heat shock response . By contrast, the groESL3 genes are co-regulated together with symbiotic nitrogen fixation genes, in that they are activated by the nitrogen fixation regulatory protein NifA at low oxygen conditions and transcribed from a -24/-12 promoter by the sigma 54 RNA polymerase . Two other members of the groESL gene family are apparently expressed constitutively at different levels, and yet another one is strongly induced by high temperature . As an attractive hypothesis it follows that B . japonicum may modulate its cellular contents of GroES- and GroEL-like chaperonins in response to specific environmental conditions and physiological needs.

C R Acad Sci III, 1993 Jul, 316(7), 661 - 6
Uptake of iron from ferric-citrate in the cyanobacteria Synechocystis PCC6803; Laboure AM et al.; Iron is an essential element for bacterial growth . Its intracellular concentration is mainly controlled at the uptake level, which is mediated by iron-dicitrate transport in various prokaryotes . We report here that the cyanobacterium Synechocystis PCC6803 takes up iron from ferric citrate . This process is dependent of the iron:citrate ratio, with a better efficiency for the highest concentration of citrate . The iron-citrate complex is the substrate of this active uptake, inhibited in part by the uncoupler FCCP . Iron starvation activates this uptake . These data suggest the existence of a specific ferric citrate receptor on the cyanobacterial membrane.

Cell Mol Biol (Noisy-le-grand), 1993 Jul, 39(5), 491 - 501
Epitope mapping of human thyroid peroxidase defined seven epitopes recognized by sera from patients with thyroid pathologies; Zanelli E et al.; Human thyroid peroxidase (hTPO) is the major component of the microsomal antigen . In almost cases, antibodies against this protein are found in sera from patients with autoimmune thyroid diseases . Overlapping cDNAs which correspond to the complete hTPO mRNA obtained from a thyroid library or by polymerase chain reaction were cloned and expressed as fusion proteins in a prokaryotic vector . Seven antigenic determinants between 21 and 49 amino acids were defined by cloning, subcloning of the immunoreactive regions and screenings with the two rabbit polyclonal anti hTPO antibodies . This study confirms the antigenic nature of the sequences 70-160 and 590-675 but above all refines the localization of three shorter distinct antigenic peptides corresponding to the sequences 68-105, 106-126 and 574-621 . Moreover, four other determinants were characterized on the sequences 233-277, 467-515, 641-685 and 701-730 . Analysis of sera from patients with autoimmune thyroid diseases (AITD) against the seven immunoreactive peptides confirms the heterogeneous nature of autoantibodies to hTPO.

EMBO J, 1993 Jul, 12(7), 2617 - 24
Crystal structure of the SH3 domain in human Fyn; comparison of the three-dimensional structures of SH3 domains in tyrosine kinases and spectrin; Noble ME et al.; The Src-homology 3 (SH3) region is a protein domain consisting of approximately 60 residues . It occurs in a large number of eukaryotic proteins involved in signal transduction, cell polarization and membrane--cytoskeleton interactions . The function is unknown, but it is probably involved in specific protein--protein interactions . Here we report the crystal structure of the SH3 domain of Fyn (a Src family tyrosine kinase) at 1.9 A resolution . The crystals have two SH3 molecules per asymmetric unit . These two Fyn SH3 domains are not related by a local twofold axis . The crystal structures of spectrin and Fyn SH3 domains as well as the solution structure of the Src SH3 domain show that these all have the same basic fold . A protein domain which has the same topology as SH3 is present in the prokaryotic regulatory enzyme BirA . The comparison between the crystal structures of Fyn and spectrin SH3 domains shows that a conserved surface patch, consisting mainly of aromatic residues, is flanked by two hairpin-like loops (residues 94-104 and 114-118 in Fyn) . These loops are different in tyrosine kinase and spectrin SH3 domains . They could modulate the binding properties of the aromatic surface.

J Immunol, 1993 Jul 1, 151(1), 201 - 10
Differential amino-terminal anchors for peptide binding to H-2M3a or H-2Kb and H-2Db; Shawar SM et al.; We previously established that H-2M3a, the H chain of the maternally transmitted Ag (Mta), is specialized for presentation of N-formylated peptides . We hypothesized that the N-formyl group might prevent or limit the presentation of peptide Ag by H-2K and H-2D molecules . We now show by Mta- and OVA-specific CTL assays, peptide competition, and immunofluorescence analyses that N-formyl modification of four antigenic peptides inhibited their binding by either H-2Kb (OVAMet258-264, VSVNP52-59, and SVNP324-332) or H2-Db (SVNP324-332, and IVNP366-374) . In contrast, N-formyl-OVAMet258-264 did bind to H2-M3a . The data imply lack of an N-formyl-binding pocket in classical MHC class I molecules and are consistent with a specialized role for H2-M3a in presentation of N-formylated peptides such as derived from intracellular prokaryotic parasites.

J Biol Chem, 1993 Jun 25, 268(18), 13081 - 8
Activation of the phosphosignaling protein CheY . I . Analysis of the phosphorylated conformation by 19F NMR and protein engineering; Drake SK et al.; CheY, the 14-kDa response regulator protein of the Escherichia coli chemotaxis pathway, is activated by phosphorylation of Asp57 . In order to probe the structural changes associated with activation, an approach which combines 19F NMR, protein engineering, and the known crystal structure of one conformer has been utilized . This first of two papers examines the effects of Mg(II) binding and phosphorylation on the conformation of CheY . The molecule was selectively labeled at its six phenylalanine positions by incorporation of 4-fluorophenylalanine, which yielded no significant effect on activity . One of these 19F probe positions monitored the vicinity of Lys109, which forms a salt bridge to Asp57 in the apoprotein and has been proposed to act as a structural "switch" in activation . 19F NMR chemical shift studies of the labeled protein revealed that the binding of the cofactor Mg(II) triggered local structural changes in the activation site, but did not perturb the probe of the Lys109 region . The structural changes associated with phosphorylation were then examined, utilizing acetyl phosphate to chemically generate phsopho-CheY during NMR acquisition . Phosphorylation triggered a long-range conformational change extending from the activation site to a cluster of 4 phenylalanine residues at the other end of the molecule . However, phosphorylation did not perturb the probe of Lys109 . The observed phosphorylated conformer is proposed to be the first step in the activation of CheY; later steps appear to perturb Lys109, as evidenced in the following paper . Together these results may give insight into the activation of other prokaryotic response regulators.

Biochim Biophys Acta, 1993 Jun 25, 1173(3), 266 - 72
Binding to DNA of selected lexitropsins and effects on prokaryotic topoisomerase activity; Burckhardt G et al.; The binding behaviour toward DNA of some minor groove binders related to distamycin was studied by means of circular dichroism . In addition their influence on the activity of topoisomerases isolated from Streptomyces noursei has been investigated . The monocationic imidazole containing ligands (lexitropsins) show a decreased affinity to AT pairs but an increased affinity to GC pairs which contrasts the AT-preferred binding of Dst-2 and Dst-3 . For the monocationic triimidazole containing lexitropsin the affinity for GC over AT pairs was most pronounced . It was also found that the imidazole containing lexitropsins are inhibitors of topoisomerases . These minor groove binders interfere more strongly with the DNA gyrase activity than with the prokaryotic topoisomerase I . Our results indicate that Dst-3 most effectively inhibits gyrase and topoisomerase I activity . However, the inhibitory effect is neither related to the base pair specificity nor to the binding strength of different ligands . The mechanism of interference of minor groove binders with topoisomerase activity is more complex . It is considered that different factors, such as the nature of the ligand together with their DNA binding parameters and the target sequences of the enzymes play a role in the inhibitory effects of minor groove binders.

Nucleic Acids Res, 1993 Jun 25, 21(12), 2891 - 7
Termination of translation in bacteria may be modulated via specific interaction between peptide chain release factor 2 and the last peptidyl-tRNA(Ser/Phe); Arkov AL et al.; The 5' context of 671 Escherichia coli stop codons UGA and UAA has been compared with the context of stop-like codons (UAC, UAU and CAA for UAA; UGG, UGC, UGU and CGA for UGA) . We have observed highly significant deviations from the expected nucleotide distribution: adenine is over-represented whereas pyrimidines are under-represented in position -2 upstream from UAA . Uridine is over-represented in position -3 upstream from UGA . Lysine codons are preferable immediately prior to UAA . A complete set of codons for serine and the phenylalanine UUC codon are preferable immediately 5' to UGA . This non-random codon distribution before stop codons could be considered as a molecular device for modulation of translation termination . We have found that certain fragment of E . coli release factor 2 (RF2) (amino acids 93-114) is similar to the amino acid sequences of seryl-tRNA synthetase (positions 10-19 and 80-93) and of beta (small) subunit (positions 72-94) of phenylalanyl-tRNA synthetase from E . coli . Three-dimensional structure of E . coli seryl-tRNA synthetase is known {1}: Its N-terminus represents an antiparallel alpha-helical coiled-coil domain and contains a region homologous to RF2 . On the basis of the above-mentioned results we assume that a specific interaction between RF2 and the last peptidyl-tRNA(Ser/Phe) occurs during polypeptide chain termination in prokaryotic ribosomes.

Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5524 - 8
Interactions between three common subunits of yeast RNA polymerases I and III; Lalo D et al.; The AC40 and AC19 subunits (encoded by RPC40 and RPC19) are shared by yeast RNA polymerases I and III and have a local sequence similarity to prokaryotic alpha subunits . Mutational analysis of the corresponding "alpha motif" indicated that its integrity is essential on AC40 subunit but is not essential on AC19 subunit . By applying the two-hybrid method, these two polypeptides were shown to associate in vivo . Extragenic suppression of rpc19 and rpc40 mutations confirmed that AC19 and AC40 subunits interact with each other in vivo and revealed an interaction with ABC10 beta subunit {encoded by RPB10; Woychick, N . A . & Young, R.A . (1990) J . Biol . Chem . 265, 17816-17819}, one of the five polypeptides common to all three nuclear RNA polymerases . A correction of the RPB10 sequence showed that ABC10 beta subunit is a 70-amino acid polypeptide, as confirmed by peptide microsequencing . These results suggest that the assembly of RNA polymerase I and III requires the association of ABC10 beta subunit with an AC19/AC40 heterodimer.

Eur J Biochem, 1993 Jun 15, 214(3), 869 - 77
Yeast seryl-tRNA synthetase expressed in Escherichia coli recognizes bacterial serine-specific tRNAs in vivo; Weygand-Durasevic I et al.; The Saccharomyces cerevisiae serS gene which encodes seryl-tRNA synthetase (SerRS) was expressed in Escherichia coli from the promoter and the ribosome binding sequences contained in its own 5'-flanking region . The low level of yeast SerRS in the prokaryotic host was sufficient to permit in vivo complementation of two temperature-sensitive E . coli serS mutants at the nonpermissive temperature . Thus, yeast SerRS can aminoacylate E . coli tRNA(Ser) species in vivo . Yeast SerRS, isolated from an overexpressing E . coli strain by a rapid two-step purification on FPLC, aminoacylated E . coli tRNA with serine much more poorly (relative kcat/Km = 2 x 10(-4)) than its homologous tRNAs . DL-Serine hydroxamate, an inhibitor of E . coli SerRS, inhibits yeast SerRS in vivo and in vitro with an inhibition constant (Ki) of 2.7 mM, a value 90-fold higher than that for E . coli SerRS.

Cas Lek Cesk, 1993 Jun 14, 132(12), 353 - 8
{What is cytochrome P-450? Enzyme forms of cytochrome P-450--extent of knowledge}; Soucek P et al.; The submitted review summarizes contemporary knowledge of the enzyme P 450, the importance of which in human metabolic processes has been beyond doubt for a long time . The existence of various forms of the enzyme (there is a number of enzymatic forms of this haemoprotein which does not differ only as regards the structure of the alipoprotein but also as regards some physical and chemical properties) was proved in many prokaryotic and eukaryotic organisms . It played a special role in herbivores . New forms were detected which can be induced e . g . by polycyclic hydrocarbons, barbiturates, steroids, ethanol (1), hypolipidaemic substances and some antibiotics . Also other regulating factors are known . The clinical use of P 450 is complicated so far by the great genetically conditioned multiplicity of its forms in man . However, a number of very promising and important applications are foreseen.

Nucleic Acids Res, 1993 Jun 11, 21(11), 2541 - 7
A common set of conserved motifs in a vast variety of putative nucleic acid-dependent ATPases including MCM proteins involved in the initiation of eukaryotic DNA replication; Koonin EV; A new superfamily of (putative) DNA-dependent ATPases is described that includes the ATPase domains of prokaryotic NtrC-related transcription regulators, MCM proteins involved in the initiation of eukaryotic DNA replication, and a group of uncharacterized bacterial and chloroplast proteins . MCM proteins are shown to contain a modified form of the ATP-binding motif and are predicted to mediate ATP-dependent opening of double-stranded DNA in the replication origins . In a second line of investigation, it is demonstrated that the products of unidentified open reading frames from Marchantia mitochondria and from yeast, and a domain of a baculovirus protein involved in viral DNA replication are related to the superfamily III of DNA and RNA helicases that previously has been known to include only proteins of small viruses . Comparison of the multiple alignments showed that the proteins of the NtrC superfamily and the helicases of superfamily III share three related sequence motifs tightly packed in the ATPase domain that consists of 100-150 amino acid residues . A similar array of conserved motifs is found in the family of DnaA-related ATPases . It is hypothesized that the three large groups of nucleic acid-dependent ATPases have similar structure of the core ATPase domain and have evolved from a common ancestor.

Nucleic Acids Res, 1993 Jun 11, 21(11), 2649 - 53
A small RNA of Mycoplasma capricolum that resembles eukaryotic U6 small nuclear RNA; Ushida C et al.; Mycoplasma capricolum, a parasitic prokaryote, contains several small stable RNAs, besides rRNAs and tRNAs . One of them, designated MCS4 RNA (125 nucleotides in length), has been isolated and sequenced . This RNA is abundant in the cell, and is encoded by two genes . Unexpectedly, MCS4 RNA has been found to reveal extensive sequence similarity to eukaryotic U6 snRNAs . This finding suggests that MCS4 and U6 snRNAs are derived from a common ancestral RNA that has existed before the divergence of prokaryotes and eukaryotes.

Eur J Biochem, 1993 Jun 1, 214(2), 475 - 81
Isolation, characterization and N-terminal amino acid sequence of hydrogenase from the green alga Chlamydomonas reinhardtii; Happe T et al.; Hydrogenase from Chlamydomonas reinhardtii was purified to homogeneity by five column-chromatography steps under strict anaerobic conditions . The cells were disrupted by mild treatment with detergent . The enzyme was purified 6100-fold, resulting in a specific activity for H2 evolution of 935 mumol.min-1.mg protein-1 at 25 degrees C, using reduced methyl viologen as electron donor . The optimal temperature for hydrogen evolution is 60 degrees C, the optimal pH value is 6.9 . The Km value for methyl viologen is 0.83 mM, for ferredoxin, 35 microM . From SDS/PAGE gels, the protein was judged to be pure . On non-denaturing gels, run under nitrogen, a single band was detected after activity staining . This band corresponded to the single band observed on denaturing SDS gels, which had an apparent molecular mass of 48 kDa . If the band was cut out of the native gel and incubated with reduced methyl viologen, hydrogen evolution could be measured . The purified enzyme contains 4 Fe atoms/mol . The amino acid composition and the N-terminal amino acid sequence (24 residues) of the protein were determined . No significant amino acid sequence homologies could be found to any sequences from prokaryotic hydrogenases.

Proc Natl Acad Sci U S A, 1993 Jun 1, 90(11), 4907 - 11
Nucleotide-excision repair of DNA in cell-free extracts of the yeast Saccharomyces cerevisiae; Wang Z et al.; A wide spectrum of DNA lesions are repaired by the nucleotide-excision repair (NER) pathway in both eukaryotic and prokaryotic cells . We have developed a cell-free system in Saccharomyces cerevisiae that supports NER . NER was monitored by measuring repair synthesis in DNA treated with cisplatin or with UV radiation . Repair synthesis in vitro was defective in extracts of rad1, rad2, and rad10 mutant cells, all of which have mutations in genes whose products are known to be required for NER in vivo . Additionally, repair synthesis was complemented by mixing different mutant extracts, or by adding purified Rad1 or Rad10 protein to rad1 or rad10 mutant extracts, respectively . The latter observation demonstrates that the Rad1 and Rad10 proteins directly participate in the biochemical pathway of NER . NER supported by nuclear extracts requires ATP and Mg2+ and is stimulated by polyethylene glycol and by small amounts of whole cell extract containing overexpressed Rad2 protein . The nuclear extracts also contain base-excision repair activity that is present at wild-type levels in rad mutant extracts . This cell-free system is expected to facilitate studies on the biochemical pathway of NER in S . cerevisiae.

Biochem J, 1993 Jun 1, 292 ( Pt 2), 571 - 6
Over-expression of a functionally active human GM2-activator protein in Escherichia coli; Klima H et al.; The cDNA of the human GM2-activator protein was cloned into the expression vector pHX17 . The plasmid encodes a fusion protein with a hexahistidine tail and a Factor Xa cleavage site at its N-terminus . The recombinant protein was purified from cell homogenates under denaturing conditions by metal-ion affinity chromatography in a single step and then was refolded . The hexahistidine tail could be removed when desired by digestion with Factor Xa . In a functional assay, the GM2-activator thus generated from Escherichia coli and renatured, with or without the hexahistidine tail, was as active as the native GM2-activator protein that was purified from human tissue . When added to the culture medium, the recombinant carbohydrate-free GM2-activator, carrying the hexahistidine tail, could be taken up efficiently and restored the degradation of ganglioside GM2 to normal rates in mutant fibroblasts with the AB variant of GM2-gangliosidosis, which is characterized by a genetic defect in the GM2-activator protein . The prokaryotic expression system is useful for producing milligram quantities of a pure and functionally active GM2-activator.

Trop Med Parasitol, 1993 Jun, 44(2), 116 - 8
Analysis of the genomic sequence encoding the 29-kDa cysteine-rich protein of Entamoeba histolytica; Bruchhaus I et al.; By analyzing cDNA and genomic clones coding for the 29-kDa cysteine-rich protein of Entamoeba histolytica, substantial sequence differences were found to the 5'-end of a previously described full-length cDNA coding for the same protein (Reed et al., 1992), which was reported to contain an untranslated 5'-sequence of at least 171 nucleotides, unusual for E . histolytica cDNAs . We found evidence that the cDNA published by Reed et al . (1992) represents a hybridclone composed of two unrelated sequences and that the gene coding for the 29-kDa molecule comprises all of the features typical for E . histolytica genes . A data base analysis revealed substantial sequence homology of the 29-kDa protein to a class of polypeptides found in prokaryotic organisms that may be involved in the inactivation of hydrogen peroxide.

Mol Microbiol, 1993 Jun, 8(6), 1115 - 24
Isolation and analysis of a linear plasmid-located gene of Borrelia burgdorferi B29 encoding a 27 kDa surface lipoprotein (P27) and its overexpression in Escherichia coli; Reindl M et al.; Using an antiserum of a patient with cutaneous manifestations of Lyme borreliosis we have isolated the gene encoding a 27 kDa protein antigen (P27) of Borrelia burgdorferi B29 from a lambda-gt11 expression library . Nucleotide sequence analysis revealed that it is a basic protein of 248 amino acids with a typical prokaryotic leader sequence of 17 amino acid residues at the N-terminus of the proposed translation product . Biochemical investigations showed that P27 is a surface-exposed lipoprotein . From pulsed-field gel electrophoresis and subsequent Southern blot analysis it is evident that the p27 gene is located on a linear plasmid of a size of approximately 55 kb . It was overexpressed in Escherichia coli and the purified recombinant protein was used for biochemical and serological studies . Northern and Western blot analysis demonstrated that p27 is expressed in the European B . burgdorferi strain B29, but not in the American strain B31.

Yeast, 1993 Jun, 9(6), 669 - 75
Para-aminobenzoate synthase gene of Saccharomyces cerevisiae encodes a bifunctional enzyme; Edman JC et al.; The Saccharomyces cerevisiae gene for para-aminobenzoate (PABA) synthase has been identified based upon its ability to confer sulfonamide resistance when present on a multicopy episomal vector . The 3840 bp DNA sequence fragment reported here contains a 2199 bp open reading frame encoding a 733 amino acid protein with similarity to the two components of PABA synthase described for prokaryotes (Escherichia coli PabA and PabB), suggesting that PABA synthase is bifunctional in yeast . The cloned sequence was confirmed to be PABA synthase by gene disruption . Chromosome gel analysis places the gene for PABA synthase on chromosome XIV.

Trends Biochem Sci, 1993 Jun, 18(6), 202 - 6
Multimeric complexes formed by DNA-binding proteins of low sequence specificity; Serrano M et al.; Some proteins bind to double-stranded DNA with low sequence specificity, forming regular multimeric complexes that extend over large regions of DNA, strongly distorting its conformation . Formation of these complexes at particular DNA sites usually depends on the structural ability of the DNA to follow the path imposed by the protein array . These complexes are found in both prokaryotic and eukaryotic organisms and participate in processes such as DNA replication, transcription and packaging.

Semin Arthritis Rheum, 1993 Jun, 22(6), 357 - 74
Heat shock (stress) proteins and autoimmunity in rheumatic diseases; Schultz DR et al.; The rheumatic diseases (RDs) are characterized by acute and chronic inflammation, and autoimmunity plays a major role in their pathogenesis . RDs are for the most part of unknown etiology, but recent evidence indicates that heat shock or stress proteins (HSPs) may have an important role in the etiology/pathogenesis of RDs . HSPs are produced by prokaryotic and eukaryotic cells and are grouped according to molecular weight . Phylogenetically, HSPs are very old and are remarkably conserved molecules in evolution from bacteria to humans . HSPs are induced by a variety of cellular stresses in addition to heat; cognates are expressed constitutively and are essential in a number of normal functions . Some HSPs serve as molecular chaperones, the latter defined as proteins that mediate folding of other polypeptides and either promote their assembly into oligomeric structures or disassemble the final product . Conservation of structure and function of many HSPs may provide a link between immunity to infection and the autoimmune features of RDs . Evidence is reviewed from clinical and laboratory observations that diverse microbial agents, including viruses, bacteria, and parasites, may have putative roles in the development and pathogenesis of some RDs . HSPs also are discussed in relation to the major histocompatibility complex, HLA antigens, and disease associations and how they may alter the balance between tolerance and autoimmunity . Studies are reviewed that are supportive or nonsupportive of the concept of microbial infection associated with autoimmunity; individuals first react to microbial immunizations or infections with enhanced cellular/humoral responses to the agent's HSPs . With the enhanced immune response, cross-reactivity may occur with an HSP of the stressed host because of structural similarities to the microbial HSP . If all of these events occur, the host's homologous HSP or stressed cells now become true autoantigen(s) . This sequence has implications for the etiology of immune-mediated RDs, the concept of epitope sharing, and the accompanying autoimmunity . A recurring theme emphasized in some reports to understand better the role of HSPs in autoimmunity is the need to select patients with early-onset disease . A minor subpopulation of T lymphocytes express a CD3-associated T-cell receptor (TCR) heterodimer composed of gamma and delta polypeptide chains . The gamma delta + T cells have several unique features . When analyzed by the polymerase chain reaction, lymphocytes with TCR-gamma delta appear to reflect the polyclonal expansion of preexisting gamma delta clones . They are found in peripheral lymphoid tissue in very low percentage (< 5%) but may represent the majority of T cells within epithelial tissue.(ABSTRACT TRUNCATED AT 400 WORDS)

Genetics, 1993 Jun, 134(2), 401 - 8
The effect of DNA sequence divergence on sexual isolation in Bacillus; Roberts MS et al.; We have investigated the relationship between sexual isolation and DNA sequence divergence in the transformation (at locus rpoB) of a naturally competent strain of Bacillus subtilis . Using both genomic DNA and a PCR-amplified segment of gene rpoB as donor, we found that the extent of sexual isolation at locus rpoB was closely predicted, over three orders of magnitude, as a log-linear function of sequence divergence at that locus . Because sexual isolation between a recipient and any potential donor may be determined as a general mathematical function of sequence divergence, transformation is perhaps the only sexual system, in either the prokaryotic or the eukaryotic world, in which sexual isolation can be predicted for a pair of species without having to perform the cross . These observations suggest the possibility of a general approach to the indirect prediction of sexual isolation in bacteria recombining principally by natural transformation.

Mol Ecol, 1993 Jun, 2(3), 171 - 81
Plasmid DNA in a groundwater aquifer microcosm--adsorption, DNAase resistance and natural genetic transformation of Bacillus subtilis; Romanowski G et al.; Prokaryotes can exchange chromosomal and plasmid genes via extracellular DNA in a process termed genetic transformation . This process has been observed in the test tube for several bacterial species living in the environment but it is not clear whether transformation occurs in natural bacterial habitats . A major constituent of terrestrial environments are solid particles such as quartz, silt and clay, which have considerable surface areas and which make up the solid-liquid interfaces of the habitat . In previous experiments the adsorption of DNA to chemically purified quartz and clay minerals was shown and the partial protection of adsorbed DNA against DNAase I . In a microcosm consisting of natural groundwater aquifer material (GWA) sampled directly from the environment and groundwater (GW) both linear duplex and supercoiled plasmid DNA molecules bound rapidly and quantitatively to the minerals . The divalent cations required to form the association were those present in the GWA/GW microcosm . The association was stable to extended elution over one week at 23 degrees C . Upon adsorption, the DNA became highly resistant against enzymatic degradation . About 1000 times higher DNAase I concentrations were needed to degrade bound DNA to the same extent as DNA dissolved in GW . Furthermore, chromosomal and plasmid DNA bound on GWA transformed competent cells of Bacillus subtilis . However, in contrast to DNA in solution, on GWA the chromosomal DNA was more active in transformation than the plasmid DNA . The studies also revealed that in the transformation of B . subtilis Mg2+ can be replaced by Na+, K+ or NH4+ . The observations suggest that in soil and sediment environments, mineral material with inorganic precipitates and organic matter can harbour extracellular DNA leaving it available for genetic transformation.

J Mol Evol, 1993 Jun, 36(6), 568 - 77
Occurrence of chaperonin 60 and chaperonin 10 in primary and secondary bacterial symbionts of aphids: implications for the evolution of an endosymbiotic system in aphids; Fukatsu T et al.; All aphids harbor symbiotrophic prokaryotes ("primary symbionts") in a specialized-abdominal cell, the bacteriocyte . Chaperonin 60 (Cpn60, symbionin) and chaperonin 10 (Cpn10), which are high and low molecular weight heatshock proteins, were sought in tissues of more than 60 aphid species . The endosymbionts were compared immunologically and histologically . It was demonstrated that (1) there are two types of aphids in terms of the endosymbiotic system: some with only primary symbionts and others with, in addition, secondary symbionts; (2) the primary symbionts of various aphids are quite similar in morphology whereas the secondary symbionts vary; and (3) irrespective of the aphid species, Cpn60 is abundant in both the primary and secondary symbionts, while Cpn10 is abundant in the secondary symbionts but present in small amounts in the primary ones . Based on these results, we suggest that the primary symbionts have been derived from a prokaryote that was acquired by the common ancestor of aphids whereas the secondary symbionts have been acquired by various aphids independently after divergence of the aphid species . In addition, we point out the possibility that the prokaryotes under intracellular conditions have been subject to some common evolutionary pressures, and as a result, have come to resemble cell organelles.

J Gen Virol, 1993 Jun, 74 ( Pt 6), 1115 - 24
Expression of non-conserved regions of the S genome segments of three hantaviruses: evaluation of the expressed polypeptides for diagnosis of haemorrhagic fever with renal syndrome; Wang M et al.; Haemorrhagic fever with renal syndrome (HFRS) is a serious and often fatal disease caused by viruses in the Hantavirus genus of the family Bunyaviridae . We expressed the entire coding region of the small (S) genome segments of three serologically distinct hantaviruses as soluble proteins in Escherichia coli and evaluated the expressed nucleocapsid proteins (NPs) as antigens for diagnosis of HFRS . We also prepared novel diagnostic antigens by expressing truncated genes from which we deleted amino acid coding regions that were highly conserved among the three viruses . These antigens were analysed for their potential to detect and differentiate between antisera to various hantaviruses by ELISA . ELISA results obtained with HFRS patient sera or with sera from naturally or experimentally infected animals indicate that homologous antigens and antisera reacted to high titre . The truncated NPs were more specific than the complete NPs in distinguishing between possible aetiological agents of HFRS . Our findings demonstrate that prokaryotic expression of portions of the NPs of specific hantaviruses can be used to generate, readily and efficiently, large quantities of antigen that is both sensitive and specific in diagnostic assays for HFRS.

C R Acad Sci III, 1993 Jun, 316(6), 547 - 9
{From repressor to aggregulate}; Jacob F; In eukaryotic organisms as in prokaryotes, gene expression is controlled mainly at the transcription level . In many cases, this regulation is performed by aggregates of transcription factors for which the name aggregulate is proposed.

Antiviral Res, 1993 Jun, 21(2), 155 - 71
The use of ampligen alone and in combination with ganciclovir and coumermycin A1 for the treatment of ducks congenitally-infected with duck hepatitis B virus; Niu J et al.; Ampligen, a known immunomodulator and interferon inducer, was used alone and in combination with other antiviral agents to treat ducks congenitally-infected with duck hepatitis B virus . These antiviral agents included the conventional nucleoside analogue ganciclovir and the prokaryotic DNA gyrase B inhibitor coumermycin A1 . When used alone, ampligen decreased the amount of serum and liver viral DNA, but had no effect on circulating duck hepatitis B surface antigen (DHBsAg) . In combination with ganciclovir, the antiviral effect appeared at least additive with a greater inhibition of viral DNA replication within the liver . The combination of ampligen with coumermycin A1 also resulted in inhibition of viral replication but to a lesser extent than ampligen alone . When all three agents were used together, viral DNA replication was again inhibited, but as with previous treatment regimes, serum DHBsAg levels remained unchanged . At the end of the treatment period for all regimes, analysis of viral DNA forms in the liver showed that the viral relaxed circular and supercoiled DNA forms had persisted . Within 1 week of cessation of therapy, viral replication had often returned to pre-treatment levels . Interferon-like activity was detected in the sera of the majority of the treated ducks during the ampligen therapy, but no clear relationship between the presence of interferon and antiviral effect could be established . These observations in the duck hepatitis B model may provide a rational basis for the use of combinations of antiviral and immunomodulatory regimes for the management of chronic hepatitis B infection in man.

DNA Cell Biol, 1993 Jun, 12(5), 441 - 53
A multifunctional prokaryotic protein expression system: overproduction, affinity purification, and selective detection; Kroll DJ et al.; A series of plasmid vectors, pRSET A, B, and C, have been developed for high-level protein expression in prokaryotes and have been characterized . Based upon the T7 RNA polymerase-driven pET system, the pRSET vectors encode recombinant proteins as fusions with a multifunctional leader peptide containing a hexahistidyl sequence for purification on Ni(2+)-affinity resins, a tyrosine residue for radioiodination, and an enterokinase proteolytic cleavage site for leader peptide removal . Monoclonal antibodies (MAbs) to two epitopes on the leader peptide, which also contains amino acids 1-12 of the T7 gene 10 major capsid protein, were developed and provide for universal immunological detection of pRSET-expressed fusion proteins . Subcloning of protein-encoding DNA is facilitated by an 11-site polylinker which is offset for all three ribosomal reading frames, and an f1(+) origin of DNA replication permits single-stranded DNA synthesis for site-directed mutagenesis protocols . Representative fusion proteins overexpressed in Escherichia coli were successfully purified under both denaturing and nondenaturing conditions by single-step Ni2+ affinity chromatography . Purification was independent of recombinant protein solubility in sonicated or freeze-thawed E . coli lysates . Isolation of MAbs for selective recognition of either of two leader peptide epitopes was demonstrated by immunoprecipitation, but this selectivity was less evident under conditions for Western blotting . In combining the utility of T7 RNA polymerase-directed expression with several recent advances in protein purification and detection, the pRSET vectors will serve as a powerful resource for a variety of studies in protein biochemistry.

J Biol Chem, 1993 May 25, 268(15), 11050 - 6
A human cDNA corresponding to a gene overexpressed during cell proliferation encodes a product sharing homology with amoebic and bacterial proteins; Prosperi MT et al.; A clone, designated pag, was isolated by differential screening of cDNA libraries made from the untransformed and ras-transformed human mammary epithelial cell line HBL100 . This cDNA corresponds to a gene constitutively expressed in most human cells which is induced to higher levels upon serum stimulation in untransformed and ras-transformed HBL100 cells . However, the abundance of the pag transcript is approximately 3-fold higher in transformed as compared to untransformed cells after 7-15 h of serum stimulation . In the promyelocytic leukemia cell line HL60 induced to differentiate the level of pag mRNA starts to decrease between 48 and 72 h following induction . During this period, which represents the commitment phase of differentiation, HL60 cells cease to proliferate . Therefore, in HBL100 and HL60 cells, higher levels of pag gene expression are correlated with cell proliferation . The pag cDNA codes for a 22-kDa protein, devoid of known consensus motifs, and shares 66% homology with a murine gene product (MER5) that is preferentially expressed in erythroleukemia cells during the early period of cell differentiation . In addition, the pag gene product shares approximately 50% identity with a 29-kDa surface antigen of Entamoeba histolytica and a 26-kDa antigen of Helicobacter pylori . Distant relationship was also found with other prokaryotic proteins . The pag cDNA hybridizes to multiple sequences within human and other mammalian genomes and to fewer sequences in chicken and Saccharomyces cerevisiae . Although a true relationship between eukaryotic and prokaryotic genes is difficult to establish, the conservation of pag gene sequences throughout Eukaryotae rather suggests that the pag locus belongs to a new class of genes encoding highly conserved proteins.

Nucleic Acids Res, 1993 May 25, 21(10), 2383 - 8
Isolation and identification by sequence homology of a putative cytosine methyltransferase from Arabidopsis thaliana; Finnegan EJ et al.; A plant cytosine methyltransferase cDNA was isolated using degenerate oligonucleotides, based on homology between prokaryote and mouse methyltransferases, and PCR to amplify a short fragment of a methyltransferase gene . A fragment of the predicted size was amplified from genomic DNA from Arabidopsis thaliana . Overlapping cDNA clones, some with homology to the PCR amplified fragment, were identified and sequenced . The assembled nucleic acid sequence is 4720 bp and encodes a protein of 1534 amino acids which has significant homology to prokaryote and mammalian cytosine methyltransferases . Like mammalian methylases, this enzyme has a C terminal methyltransferase domain linked to a second larger domain . The Arabidopsis methylase has eight of the ten conserved sequence motifs found in prokaryote cytosine-5 methyltransferases and shows 50% homology to the murine enzyme in the methyltransferase domain . The amino terminal domain is only 24% homologous to the murine enzyme and lacks the zinc binding region that has been found in methyltransferases from both mouse and man . In contrast to mouse where a single methyltransferase gene has been identified, a small multigene family with homology to the region amplified in PCR has been identified in Arabidopsis thaliana.

J Biol Chem, 1993 May 25, 268(15), 11463 - 9
Sequences in the 3'-untranslated region of the human cellular glutathione peroxidase gene are necessary and sufficient for selenocysteine incorporation at the UGA codon; Shen Q et al.; Glutathione peroxidase (EC 1.11.1.9) is one of a unique group of prokaryotic and eukaryotic enzymes that contain the unusual amino acid selenocysteine . The genes for these selenoproteins encode for the atypical amino acid at a TGA codon (UGA in the mRNA transcripts), which normally functions as a termination signal . The present studies analyzed the functional importance of sequences in the coding and 3'-untranslated regions of transcripts of the primary human cellular glutathione peroxidase gene (GPX1) to the insertion of selenocysteine at this UGA codon . Deletions in potential stem-loop or hairpin structures in the coding region did not substantially diminish incorporation of selenocysteine into glutathione peroxidase transiently expressed by the pCMV4 vector in COS-1 cells . However, selenocysteine insertion was completely abolished by deletion of four-nucleotide sequences in the 3'-untranslated region from within a conserved "selenocysteine insertion sequence" motif also found in the 3'-untranslated region of mammalian genes for other selenoproteins . Moreover, in constructs fusing the glutathione peroxidase 3'-untranslated region to the coding region of rab5b (an unrelated protein normally without any selenium moiety), the glutathione peroxidase 3'-untranslated region was sufficient to direct the translation of an opal (UGA) mutation as selenocysteine . Thus, our data directly demonstrate the importance of the selenocysteine insertion motif in the glutathione peroxidase gene and specifically show that sequence elements in the 3'-untranslated region are both necessary and sufficient for translational insertion of selenocysteine at a UGA codon in eukaryotic mRNA.

Biochim Biophys Acta, 1993 May 20, 1168(1), 94 - 9
Biosynthesis of eukaryotic lipid molecular species by the cyanobacterium Spirulina platensis; Quoc KP et al.; This report brings evidence that a prokaryotic photosynthetic organism can synthesize eukaryotic molecular species of glycerolipids . When Spirulina platensis PCC 8005 was supplemented with oleic acid, the sum of the percentages of 18 carbon (C18) fatty acids in monogalactosyldiacylglycerol (MGDG), the major lipid class, became largely higher than 50 mol% . This was absolutely unexpected from the well-known structure of cyanobacterial lipids . In these organisms, C18 fatty acids usually account for less than 50 mol% because they are esterified on carbon 1 of the glycerol, exclusively . This classical feature was 99% confirmed in control as well as in palmitate-supplemented cultures . The major molecular species of MGDG, which resulted from the different distributions of fatty acids on carbons 1 and 2 of glycerol, were C18/C16 type, belonging to the so-called "prokaryotic" type of lipids . By contrast, the molecular species of MGDG from oleate-supplemented cultures consisted of only 74 mol% of C18/C16 and the complementary 26 mol% were C18/C18, the so-called "eukaryotic" type of lipids . Furthermore, such C18/C18 lipids were also evidenced as traces (< 1%) in control cultures . These results underline the fact that the fatty acid specificity of 1-monoacylglycerol-3-phosphate-acyltransferase (in Spirulina) is not as absolute as the widely accepted concept of prokaryotic lipid would suggest . Oleate, supplemented at high concentration, can be compelled to act as a substrate for the acyltransferase and this results in the appearance of C18/C18 "eukaryotic" lipids in a prokaryotic organism.

J Mol Biol, 1993 May 20, 231(2), 335 - 42
Requirement of both kinase and phosphatase activities of an Escherichia coli receptor (Taz1) for ligand-dependent signal transduction; Yang Y et al.; Taz1 is a hybrid receptor in the Escherichia coli cytoplasmic membrane, consisting of the N-terminal ligand binding domain of Tar (a chemoreceptor for aspartate) and the C-terminal signaling domain of EnvZ (an osmosensor) . The binding of aspartate to an extra cytoplasmic domain induces the transmembrane signal to the cytoplasmic signaling domain . The signaling domain functioning as a protein kinase evokes a response by transferring a phosphate from an intracellular histidine to OmpR . This domain also encodes an OmpR-specific phosphatase whose action is crucial in completing the OmpR phosphorylation cycle . Phosphorylated OmpR acts as a transcriptional activator for the ompC gene . A number of mutations were introduced into the signaling domain in conserved sequences of the prokaryotic histidine kinase family . All Taz1 mutants lost the ability to both autophosphorylate the histidine residue and transfer the phosphate to OmpR . These mutated receptors were unable to activate ompC-lacZ expression . However, ompC-lacZ was able to be activated by complementation of Taz1 mutants . In some combinations, two different defective Taz1 mutants could restore both OmpR kinase and phosphatase activities when co-expressed . In other combinations only kinase activity was restored . Aspartate-inducible ompC-lacZ expression was restored only in the former cases, while in the latter cases ompC-lacZ expression became constitutive . These results indicate that the kinase activity is essential to activate ompC expression while the phosphatase activity is required to regulate ompC gene expression in a ligand-dependent manner.

FEMS Microbiol Lett, 1993 May 15, 109(2-3), 173 - 8
Effect of different positively charged amino acids, C-terminally of the signal peptidase cleavage site, on the translocation kinetics of a precursor protein in Escherichia coli K-12; Struyve M et al.; Introduction of positively charged amino acids immediately downstream of the signal sequence in prokaryotic precursor proteins is known to affect the export process . However, it is not clear whether different positively charged amino acids affect the export process similarly . To investigate this, the glutamate at position +2 of outer membrane protein PhoE was substituted by arginine, lysine or histidine . Pulse-chase experiments revealed that the Lys and Arg residues at position +2 caused a reduced processing rate, and that the effect was markedly more severe in the case of the Arg residue . Trypsin accessibility experiments revealed that the accumulated precursors were present in the cytoplasm . Since the degree of the inhibitory effect corresponded to the pKa of the different positively charged amino acids, this suggests that the positively charged residues must be deprotonated during the secretory process.

Biochem Biophys Res Commun, 1993 May 14, 192(3), 1327 - 33
Convenient plasmid for extrachromosomal DNA recombination in mouse cells; Kameyama K et al.; We constructed a convenient plasmid for DNA recombination assay . The plasmid, pMR1, contains a double prokaryotic terminator to decrease the background and two unique restriction enzyme sites on both sides of the double terminator to allow for easy construction . The assay is capable of selecting the bacterial cells containing recombined plasmid DNA on a selective plate containing ampicillin and chloramphenicol . We adapted pMR1 for V(D)J recombination and homologous recombination and detected both types of recombination in murine PreB cell line . As pMR1 has the double terminator, background on the selective plate decreases effectively and we select only the recombined clones . We consider the vector, pMR1, to be convenient for the analysis of homologous and non-homologous recombinations.

Cell, 1993 May 7, 73(3), 469 - 85
Natural resistance to infection with intracellular parasites: isolation of a candidate for Bcg; Vidal SM et al.; Natural resistance to infection with intracellular parasites is controlled by a dominant gene on mouse chromosome 1, called Bcg, Lsh, or Ity . Bcg affects the capacity of macrophages to destroy ingested intracellular parasites early during infection . We have assembled a 400 kb bacteriophage and cosmid contig within the genomic interval containing Bcg . A search for transcription units by exon amplification identified six novel genes in this contig . RNA expression studies showed that one of them, designated Nramp, was expressed exclusively in macrophage populations from reticuloendothelial organs and in the macrophage line J774A . Nramp encodes an integral membrane protein that has structural homology with known prokaryotic and eukaryotic transport systems, suggesting a macrophage-specific membrane transport function . Susceptibility to infection (Bcgs) in 13 Bcgr and Bcgs strains tested is associated with a nonconservative Gly-105 to Asp-105 substitution within predicted transmembrane domain 2 of Nramp.

Mol Biochem Parasitol, 1993 May, 59(1), 41 - 8
Small subunit ribosomal RNA+ of Hexamita inflata and the quest for the first branch in the eukaryotic tree; Leipe DD et al.; A phylogenetic analysis of the small subunit ribosomal RNA (16S-like rRNA) coding region from Hexamita inflata demonstrates that parasitism alone cannot explain early diverging eukaryotic lineages . Parasitic and free-living diplomonads, as well as trichomonads and microsporidia, diverge at the base of the eukaryotic tree . The relative branching order of diplomonads, trichomonads and microsporidia is influenced by outlying prokaryotic taxa with different G+C compositions in their rRNA coding regions . The high G+C prokaryotes position Giardia lamblia at the base of the eukaryotic tree but split diplomonads into a paraphyletic group . When the outlying groups are restricted to rRNAs with nominal G+C compositions, diplomonads form a monophyletic group that diverged after the microsporidia and trichomonads . This unstable branching pattern correlates with unusual nucleotide compositions in the rRNAs of G . lamblia (75% G+C) and Vairimorpha necatrix (35% G+C) . In contrast, the 51% G+C composition of the H . inflata rRNA is typical of other eukaryotic rRNAs . Its divergence after trichomonads is strongly supported by bootstrap replicates in distance analyses that do not include G . lamblia . Because of a low G+C composition in its rRNA coding region, the phylogenetic placement of V . necatrix is uncertain and the identity of the deepest branching eukaryotic lineage is ambiguous.

Mol Biochem Parasitol, 1993 May, 59(1), 155 - 66
Expression of Plasmodium falciparum lactate dehydrogenase in Escherichia coli; Bzik DJ et al.; A Plasmodium falciparum gene is described which encodes lactate dehydrogenase activity (P . falciparum LDH) . The P . falciparum LDH gene contains no introns and is present in a single copy on chromosome 13 . P . falciparum LDH was expressed in all asexual blood stages as a 1.6-kb mRNA . The predicted 316 amino acid protein coding region of P . falciparum LDH was inserted into the prokaryotic expression vector pKK223-3 and a 33-kDa protein having LDH activity was synthesized in Escherichia coli . P . falciparum LDH primary structure displays high amino acid similarity (50-57%) to vertebrate and bacterial LDH, but lacks the amino terminal extension observed in all vertebrate LDH . The majority of amino acid residues implicated in substrate and coenzyme binding and catalysis of other LDH are well conserved in P . falciparum LDH . However, several notable differences in amino acid composition were observed . P . falciparum LDH contained several distinctive single amino acid insertions and deletions compared to other LDH enzymes, and most remarkably, it contained a novel insertion of 5 amino acids within the conserved mobile loop region near arginine residue 109, a residue which is known to make contact with pyruvate in the ternary complex of other LDH . These results suggest that novel features of P . falciparum LDH primary structure may be correlated with previously characterized and distinctive kinetic, biochemical, immunochemical, and electrophoretic properties of P . falciparum LDH.

Genetics, 1993 May, 134(1), 57 - 62
Instability of a plasmid-borne inverted repeat in Saccharomyces cerevisiae; Henderson ST et al.; Inverted repeated DNA sequences are common in both prokaryotes and eukaryotes . We found that a plasmid-borne 94 base-pair inverted repeat (a perfect palindrome of 47 bp) containing a poly GT sequence is unstable in S . cerevisiae, with a minimal deletion frequency of about 10(-4)/mitotic division . Ten independent deletions had identical end points . Sequence analysis indicated that all deletions were the result of a DNA polymerase slippage event (or a recombination event) involving a 5-bp repeat (5' CGACG 3') that flanked the inverted repeat . The deletion rate and the types of deletions were unaffected by the rad52 mutation . Strains with the pms1 mutation had a 10-fold elevated frequency of instability of the inverted repeat . The types of sequence alterations observed in the pms1 background, however, were different than those seen in either the wild-type or rad52 genetic backgrounds.

Mol Gen Genet, 1993 May, 239(1-2), 251 - 6
Positively charged amino acids placed next to a signal sequence block protein translocation more efficiently in Escherichia coli than in mammalian microsomes; Johansson M et al.; Positively charged amino acids are known efficiently to block protein secretion in Escherichia coli, when placed within a short distance downstream of a signal sequence . It is not known whether the same applies to protein secretion in eukaryotic cells, though statistical studies of signal sequences of prokaryotic and eukaryotic secretory proteins have suggested that the situation may be different in this case . Here, we show that identical charge mutations in a model protein have different effects on membrane translocation in E . coli and in mammalian microsomes, and that the 'charge block' effect is much more pronounced in the prokaryotic system . This finding has implications not only for our understanding of the mechanisms of protein secretion, but also points to a potential problem in the expression of eukaryotic secretory proteins in bacteria.

J Virol, 1993 May, 67(5), 2503 - 12
A poxvirus-encoded uracil DNA glycosylase is essential for virus viability; Stuart DT et al.; Infection of cultured mammalian cells with the Leporipoxvirus Shope fibroma virus (SFV) causes the induction of a novel uracil DNA glycosylase activity in the cytoplasms of the infected cells . The induction of this activity, early in infection, correlates with the early expression of the SFV BamHI D6R open reading frame which possesses significant protein sequence similarity to eukaryotic and prokaryotic uracil DNA glycosylases . The SFV BamHI D6R open reading frame and the homologous HindIII D4R open reading frame from the Orthopoxvirus vaccinia virus were cloned under the regulation of a phage T7 promoter and expressed in Escherichia coli as insoluble high-molecular-weight aggregates . During electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, the E . coli-expressed proteins migrate with an apparent molecular mass of 25 kDa . The insoluble protein aggregate generated by expression in E . coli was solubilized in urea and, following a subsequent refolding step, displayed the ability to excise uracil residues from double-stranded plasmid DNA substrates, with the subsequent formation of apyrimidinic sites . The viral enzyme, like all other characterized uracil DNA glycosylases, is active in the presence of high concentrations of EDTA, is substrate inhibited by uracil, and does not display any endonuclease activity . Attempts to inactivate the HindIII D4R gene of vaccinia virus by targeted insertion of a dominant xanthine-guanine phosphoribosyltransferase selection marker or direct insertion of a frame-shifted oligonucleotide were uniformly unsuccessful demonstrating that, unlike the uracil DNA glycosylase described for herpesviruses, the poxvirus enzyme is essential for virus viability.

Photochem Photobiol, 1993 May, 57(5), 905 - 21
Nucleotide excision repair; Sancar A et al.; Nucleotide excision repair is the major DNA repair mechanism in all species tested . This repair system is the sole mechanism for removing bulky adducts from DNA, but it repairs essentially all DNA lesions, and thus, in addition to its main function, it plays a back-up role for other repair systems . In both pro- and eukaryotes nucleotide excision is accomplished by a multisubunit ATP-dependent nuclease . The excision nuclease of prokaryotes incises the eighth phosphodiester bond 5' and the fourth or fifth phosphodiester bond 3' to the modified nucleotide and thus excises a 12-13-mer . The excision nuclease of eukaryotes incises the 22nd, 23rd, or 24th phosphodiester bond 5' and the fifth phosphodiester bond 3' to the lesion and thus removes the adduct in a 27-29-mer . A transcription repair coupling factor encoded by the mfd gene in Escherichia coli and the ERCC6 gene in humans directs the excision nuclease to RNA polymerase stalled at a lesion in the transcribed strand and thus ensures preferential repair of this strand compared to the nontranscribed strand.

Can J Microbiol, 1993 May, 39(5), 473 - 9
The second messenger cyclic 3',5'-adenosine monophosphate in pathogenic microorganisms with special reference to protozoa; De Castro SL et al.; There is a growing body of information on signal transduction components in microorganisms . Elements of the cyclic 3',5'-adenosine monophosphate signaling system and molecules similar to hormones and receptors have been identified in the majority of prokaryotes and unicellular eukaryotes that have been studied . The presence of ligand- and receptor-like molecules in parasitic microorganisms raises the possibility that these molecules may interact with host communication systems . Adrenergic control of proliferation, differentiation, and infectivity has been described in the flagellate protozoan Trypanosoma cruzi, the etiological agent of Chagas' disease . Interactions between host and parasitic systems could also be antibody mediated, with antigenic cross-reactivity between components of their cAMP-dependent systems . In this review, we discuss these possibilities and summarize the existing data in this area.

Mol Biochem Parasitol, 1993 May, 59(1), 15 - 27
Molecular characterization of phosphoribosylpyrophosphate synthetase from Leishmania donovani; Hendrickson N et al.; The phosphoribosylpyrophosphate synthetase (PRS) enzyme from parasitic protozoa plays a critical role in the acquisition of exogenous purine bases by providing the phosphoribosylpyrophosphate substrate for phosphoribosylation . To characterize a PRS enzyme from parasitic protozoa, the prs gene was isolated from a genomic library of Leishmania donovani DNA . A 1936-bp SalI fragment was sequenced that encompassed an open reading frame of 1113 nucleotides encoding a polypeptide of 371 amino acids and 40 787 Da . After gap alignment, the leishmanial PRS exhibited 40-42% amino acid identity with a variety of mammalian and prokaryotic PRSs . L . donovani PRS also contained an approx . 20-amino acid stretch that was highly homologous to the phosphoribosylpyrophosphate binding domains of mammalian phosphoribosyltransferase enzymes . Two prs-specific transcripts of 2.6 and 2.1 kb were detected by Northern analysis, and Southern blots of genomic DNA implied that the prs locus was not tandemly repeated in the L . donovani genome . PRS activity was detected in L . donovani extracts, and apparent Km values of approx . 30 microM and approx . 1 mM were calculated for ribose-5-phosphate and ATP, respectively . PRS was sensitive to inhibition by AMP and ADP but refractory to IMP, GMP, GTP, CTP, and UTP . The high apparent Km value of the parasite enzyme for ATP and its insensitivity to inhibition by many nucleotides suggested that kinetic differences between the L . donovani and human PRSs could provide an avenue for rational therapeutic manipulation of parasitic disease . The isolation of the L . donovani prs gene now provides an opportunity to genetically dissect the determinants responsible for the function and regulation of this indispensable enzyme of purine and pyrimidine metabolism in a genus of parasitic protozoa.

Plant J, 1993 May, 3(5), 657 - 68
The origin of the bifunctional dihydrofolate reductase-thymidylate synthase isogenes of Arabidopsis thaliana; Lazar G et al.; In most prokaryotic and eukaryotic organisms dihydrofolate reductase (DHFR) and thymidylate synthase (TS) are encoded by independent genes . Evidence is presented here that the higher plant Arabidopsis thaliana has two bifunctional DHFR-TS genes . The structure of the genes, DHFR at the amino terminus and TS at the carboxy terminus, is identical to their organization in protozoa, the only other known organisms with bifunctional genes . Sequence alignments suggest that the bifunctional genes from protozoa and higher plants may have different evolutionary origins . The positions of the introns support the complementary hypothesis that the DHFR domain of the bifunctional plant genes and the monofunctional DHFR gene of vertebrates derive from a common, intron-containing progenitor, although the structure (bifunctional or monofunctional) of the ancestral gene remains indeterminate . Comparison of the two bifunctional genes of Arabidopsis indicates that the DHFR and TS domains evolved at different rates; each following the evolutionary history of their monofunctional counterparts . In contrast to the DHFR domain, the evolution of the TS domain shows a higher level of nucleotide and amino acid sequence conservation, but a remarkable variability in the intron positions.

Clin Exp Rheumatol, 1993 May-Jun, 11 Suppl 9, S25 - 8
Heat-shock proteins and juvenile chronic arthritis; de Graeff-Meeder ER et al.; Heat-shock proteins are a category of proteins that are synthesized under stressful conditions (such as increased temperatures) both by prokaryotic and eukaryotic cells . Heat-shock proteins are a major target of the immune response and thus can be considered dominant antigens . Under physiological circumstances the response to heat-shock proteins is considered to play a role in the overall defence against bacterial infections . An aberrant immune response against heat-shock proteins may lead to autoimmunity, as illustrated by adjuvant arthritis in Lewis rats . Current evidence also points towards a role of T cell immunity against heat-shock proteins in the development of human autoimmune diseases such as juvenile chronic arthritis.

Trends Biochem Sci, 1993 May, 18(5), 177 - 81
Molecular genetics of chloroplast ribosomal proteins; Subramanian AR; Chloroplasts contain a complete translational apparatus which, in land plants, synthesizes the 80 or so polypeptides encoded by the organelle's own small genome . Recent molecular genetic studies have revealed much about the chloroplast ribosomal proteins (RPs) . Some of these proteins are encoded by the chloroplast genome and others by the nuclear genome . Many of these genes have now been cloned and characterized, including some that have no prokaryotic homologues.

Mol Microbiol, 1993 May, 8(3), 507 - 15
Mechanism of initiation of transcription by Escherichia coli RNA polymerase on supercoiled template; Mishra RK et al.; DNA supercoiling is known to influence the pattern of gene expression in prokaryotes . Thus the mechanism of transcription initiation and the topological state of the template are intimately related . Using in vitro reconstituted transcription assays, composed of purified RNA polymerase and promoters in their natural topological state, we have conducted a detailed study of transcription initiation from T7 early promoters including the following steps: the formation of ternary complexes, acquisition of rifampicin resistance, release of sigma factor and the capacity for RNA chain elongation in complexes . We determined the order of these events and the length of the transcripts when each step occurred during initiation of transcription on supercoiled templates . The length of the transcripts varied in a promoter-specific manner . Analysis of abortive products formed during the initiation showed that stronger promoters go to the elongation mode at transcript lengths shorter than that required for weaker promoters.

Mol Cell Biol, 1993 May, 13(5), 2742 - 52
Cooperative binding at a distance by even-skipped protein correlates with repression and suggests a mechanism of silencing; TenHarmsel A et al.; In this study, we examined how the Drosophila developmental control gene even-skipped (eve) represses transcription . Tissue culture cells were used to show that eve contains domains which inhibit transcriptional activators present at the Ultrabithorax (Ubx) proximal promoter when bound up to 1.5 kb away from these activators . Different portions of eve were fused to a heterologous DNA binding domain to show that three adjacent regions of eve contribute to silencing . There appear to be two mechanisms by which eve protein represses transcription . In this study, we used in vitro transcription and DNA binding experiments to provide evidence for one of these mechanisms . Repression in vitro correlates with binding of eve protein to two low-affinity sites in the Ubx proximal promoter . Occupancy of these low-affinity sites is dependent upon cooperative binding of other eve molecules to a separate high-affinity site . Some of these sites are separated by over 150 bp of DNA, and the data suggest that this intervening DNA is bent to form a looped structure similar to those caused by prokaryotic repressors . One of the low-affinity sites overlaps an activator element bound by the zeste transcription factor . Binding of eve protein is shown to exclude binding by zeste protein . These data suggest a mechanism for silencing whereby a repressor protein would be targeted to DNA by a high-affinity element, which itself does not overlap activator elements . Cooperative binding of further repressor molecules to distant low-affinity sites, and competition with activators bound at these sites lead to repression at a distance.

Biotechnology (N Y), 1993 May, 11(5), 612 - 8
Efficient bacterial export of a eukaryotic cytoplasmic cytochrome; Karim A et al.; The soluble core domain of cytochrome b5 of liver endoplasmic reticulum was appended at its amino terminus to full-length alkaline phosphatase secretory signal sequence including the ribosomal binding site . The chimeric precursor gene was placed under the transcriptional control of the native pho promoter in a prokaryotic expression vector . Induction of Escherichia coli by growth in a phosphate-limited medium resulted in abundant synthesis of cytochrome b5 as detected spectrophotometrically and by visual transformation of the bacteria to a pink color . The signal-appended cytochrome b5, but not the corresponding signal-deficient derivative, was translocated across the bacterial inner membrane and processed to yield authentic, haem-assembled cytochrome b5 within the periplasm . The eventual processing of the chimeric cytochrome b5 precursor was unusual regarding the known reaction specificity of signal peptidase . The exported, mature haemoprotein was biochemically indistinguishable from its native mammalian counterpart . At peak induction, approximately 6 mg of correctly matured cytochrome b5 per liter of culture was exported . This amount of cytochrome b5 constituted 6% (w/w) of the periplasmic protein . The appearance of the exported apo-cytochrome b5 preceded the formation of holo-protein . Thus the eukaryotic cytoplasmic protein was efficiently exported from E . coli and post-translocationally modified to generate a functional haemoprotein in the periplasm.

Mutat Res, 1993 May, 299(3-4), 211 - 8
Induction of large DNA deletions by persistent nicks: a new hypothesis; Hutchinson F; DNA deletions of more than one or two base pairs are induced frequently enough so that these form a reasonable fraction of mutations for only a few mutagens . Of these agents, some such as ionizing radiations form DNA double-strand breaks, and very large deletions are thought to result from a DNA end from one break ligating with a second break on the same DNA molecule . However, deletions of kilobase pairs and more are sometimes induced by ionizing radiation at a higher rate than can be accounted for by the numbers of double-strand breaks . Published data on induced deletions in particular Escherichia coli strains suggest a process involving a single lesion that could explain several features of large deletions: frequent occurrence in mammalian cells and scarcity in prokaryotes, nonrandom location which is perhaps associated with locations of origins of replication, and differences in the fraction of deletions among mutations in various genes . Some agents inducing deletions make single-strand nicks, not double-strand breaks, and the proposed mechanism hypothesizes that the inducing lesion is a persistent nick in one DNA strand--for example, a radiation-induced single-strand break with associated damage on the complementary strand that interferes with repair.

Biochem Biophys Res Commun, 1993 Apr 30, 192(2), 616 - 26
Mammalian mitochondrial DNA sequences can function as in vivo bacterial transcription terminators; Staub JM et al.; We have used a prokaryotic terminator identification vector, pDR721, to isolate regions from rat mitochondrial DNA (mtDNA) that can act as transcription terminators in vivo . Three independent fragments having terminator capability have been mapped to three general regions of the mitochondrial genome . Two terminators, pRMT1 and pRMT3, are found within and around the D-loop and cytochrome b gene, respectively, while the third, pRMT5, is located at the 3'-end of the 16S ribosomal RNA gene . After subcloning into host cells which carried temperature sensitive mutations in the termination factor, rho protein, galactokinase assays at the permissive and non-permissive temperatures suggested that pRMT3 acted as a rho-independent termination element while the other two, pRMT1 and pRMT5, were dependent on rho protein (or a rho-like protein) for efficient transcription termination.

Gene, 1993 Apr 30, 126(2), 243 - 6
Structure of the Paramecium caudatum gene encoding the B-type of the major hemoglobin component; Yamauchi K et al.; We have found the gene (Hb) encoding the B-type of the major hemoglobin (Hb) component in the ciliated protist, Paramecium caudatum, stock K33 of syngen 3 . Gene Hb, which encodes a protein consisting of 116 amino acids (aa), had the same sequence as the A-type Hb in this species, with the exception of 10 nucleotides (nt) reflecting 5 single-aa differences . The coding region was interrupted at the position between codons 62 and 63 by one short intron, which was comparable with the second intron of plant Hb in position, phase and nt sequence . The maximum alignment of the P . caudatum Hb aa sequence with the other globin sequences from prokaryotes and lower eukaryotes indicates that these globins can be classified into two groups: one consists of protist and cyanobacterial globins of about 120 aa, and the other consists of fungal and other bacterial globins of about 150 aa having a significant homology with higher eukaryotic Hbs . These proteins share more than 25% homology with one another in each group . Our finding of the P . caudatum Hb intron corresponding to the second intron of plant Hb suggests that all of the globin-encoding genes have a common origin, and that at least two groups had already existed in the globin-encoding gene before prokaryote-eukaryote radiation.

Gene, 1993 Apr 30, 126(2), 219 - 25
Cloning of human and rat cDNAs encoding the mitochondrial single-stranded DNA-binding protein (SSB); Tiranti V et al.; We have retro-transcribed and amplified by PCR the full-length cDNAs specifying the rat and human precursors of the single-stranded mitochondrial DNA (mtDNA)-binding protein (mtSSB) . Each deduced sequence is composed of a 16-amino-acid (aa) N-terminal basic pre-sequence and a mature protein (132 aa in humans and 135 aa in the rat) . The mature proteins are highly conserved among themselves and with the mtSSB from Xenopus laevis (Xl) . Moreover, three regions of the protein are similar to corresponding domains of the SSB of Escherichia coli and to the E . coli F-sex factor SSB, indicating the existence of a broad class of DNA-binding proteins with structural and functional similarities both in prokaryotes and in prokaryote-derived organelles of higher organisms.

Proc Natl Acad Sci U S A, 1993 Apr 15, 90(8), 3192 - 6
Transgenic mouse model for neurocristopathy: Schwannomas and facial bone tumors; Jensen NA et al.; We have characterized a strain of double transgenic mice with simian virus 40 large tumor antigen and prokaryotic lacZ under the control of the myelin basic protein promoter that develops spindle-cell sarcomas and osteogenic sarcomas at 5-7 months of age . Although poorly differentiated, the spindle-cell sarcomas were characterized as malignant Schwannomas based on their neural association, the presence of basal lamina, and expression of Schwann cell-specific genes . The osteogenic sarcomas were often multiple and appeared predominantly in the facial bones, less frequently in the ribs and vertebral column, and only rarely in the appendicular skeleton . Benign osteoblastic lesions were often observed adjacent to these sarcomas . Both the osteoblastic cells in the facial skeleton and Schwann cells are regarded as neural crest derivatives . The biological properties and anatomical location of these tumors suggest that they may share a common origin from the neural crest or its derivatives . R.P . Bolande {Hum . Pathol . (1974) 5, 409-429} introduced the term neurocristopathy as a unifying concept to describe such lesions arising from the neural crest or its derivatives . Cell lines established from both bone and Schwann cell tumors arising in these transgenic mice express simian virus 40 large tumor antigen mRNA as well as functional large tumor antigen . Such cell lines are potentially valuable in the search for markers that identify mammalian neural crest derivatives.

J Biol Chem, 1993 Apr 15, 268(11), 7632 - 5
A protein-tyrosine/serine phosphatase encoded by the genome of the cyanobacterium Nostoc commune UTEX 584; Potts M et al.; Protein-tyrosine phosphorylation has long been regarded as an exclusively eukaryotic phenomenon . Although some non-eukaryotes, mainly viruses, possess genes encoding protein-tyrosine kinases or protein-tyrosine phosphatases, these were probably appropriated from the eukaryotic hosts that constitute the sites of action of these enzymes . Herein we identify a gene, iphP, from the chromosome of the cyanobacterium Nostoc commune UTEX 584 that contains the His-Cys-Xaa-Ala-Gly-Xaa-Xaa-Arg sequence characteristic of known protein-tyrosine phosphatases . The expressed gene product, IphP, displayed protein-tyrosine phosphatase activity toward phosphotyrosine residues on reduced, carboxyamidomethylated, and maleylated lysozyme with optimum activity at pH 5.0 . In addition, IphP dephosphorylated the phosphoseryl groups on casein that had been phosphorylated by the cAMP-dependent protein kinase . Cell lysates of N . commune probed with antibodies to phosphotyrosine indicated the presence of a tyrosine-phosphorylated protein of M(r) approximately 85 kDa . This tyrosine-phosphorylated protein was detected in cells grown in the presence of combined nitrogen but not in nitrogen-deficient media that induces the formation of differentiated N2-fixing cells (heterocysts) . Together, these data suggest a role for protein-tyrosine phosphorylation in regulating cellular functions in this cyanobacterium . IphP is the first protein-tyrosine phosphatase to be discovered that is encoded by the chromosomal DNA of any prokaryote . Given the free-living nature of N . commune and the phylogenetic antiquity of the cyanobacteria, these findings suggest for the first time the existence of a protein-tyrosine phosphatase of genuine, unambiguous prokaryotic ancestry, thus raising fundamental questions as to the origin and role of tyrosine phosphorylation.

Nucleic Acids Res, 1993 Apr 11, 21(7), 1647 - 53
Two cDNAs from the plant Arabidopsis thaliana that partially restore recombination proficiency and DNA-damage resistance to E . coli mutants lacking recombination-intermediate-resolution activities; Pang Q et al.; Escherichia coli ruvC recG mutants lack RuvC endonuclease, which resolves crossed-strand joint molecules (Holliday junctions) formed during homologous recombination into recombinant products, and an activity (RecG) thought to partially replace RuvC . They are therefore highly deficient in homologous recombination, and sensitive to UV light and chemical DNA-damaging agents, presumably because of inability to tolerate unrepaired DNA damage by recombinational mechanisms (Lloyd, R.G . (1991) J . Bacteriol . 173:5414-5418) . We transformed these mutants with plasmids expressing cDNAs from the plant Arabidopsis thaliana . Selection for bacteria with increased resistance to methylmethanesulfonate yielded two cDNAs, designated DRT111 and DRT112 (DNA-damage-repair/toleration) . Expression of these plant cDNAs, especially DRT111, restored conjugal recombination proficiencies in ruvC and ruvC recG mutants to nearly wild-type levels . Both plant cDNAs significantly increased resistance of both mutants to UV light and several chemical DNA-damaging agents, but did not fully correct the mutant phenotypes . Drt111 activity, but not Drt112, also increased, to nearly wild-type levels, resistance of recG single mutants to UV plus mitomycin C . The predicted Drt111 and Drt112 polypeptides, 383 and 167 amino acids respectively, show no similarity with one another or with prokaryotic Holliday resolvases . Both appear chloroplast targeted; Drt112 is highly homologous to Arabidopsis plastocyanin . DRT111 and DRT112 probes hybridize only to DNA from closely related plants.

Nucleic Acids Res, 1993 Apr 11, 21(7), 1577 - 80
A chicken RAD51 homologue is expressed at high levels in lymphoid and reproductive organs; Bezzubova O et al.; Comparisons of the amino acid sequences of three yeast RecA-like proteins, Rad51 and DMC1 from S.cerevisiae and Rad51 from S.pombe, revealed several highly conserved regions . Degenerated oligonucleotides encoding two of these regions were used for the polymerase chain reaction to clone a chicken RecA-like gene . The encoded protein shares 68% and 49% identical amino acids with the Rad51 and DMC1 proteins . The strong sequence conservation between the yeast and chicken genes indicates that RecA homologues are conserved throughout evolution from prokaryotes to higher eukaryotes . High expression of the chicken Rad51 gene was found within the organs of lymphoid and germ cell development suggesting its involvement in lymphoid and meiotic recombination.

Regul Pept, 1993 Apr 8, 44(3), 305 - 9
Discussion paper: towards a systematic classification for regulatory peptides; Smart D et al.; Advances in microsequencing technology have led to the elucidation of primary structures of peptidic messengers from many eukaryotic and prokaryotic life forms . Existing peptide nomenclature is based upon such factors as bioactivity, source of isolation (tissue or species), chemical attributes, acronyms derived from a combination of these factors, or by other arbitrary means . In order to overcome many of the problems arising from current nomenclature, a standardised classification scheme for peptidic messengers is proposed which utilises an alphanumeric code string analogous to EC enzyme classification to convey information on origin, chemistry and relatedness to other similar molecules . It is anticipated that the scheme outlined will provide the basis for the rational classification of new peptidesPublication Types:

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