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T-kininogen gene expression is induced during aging.

We have constructed a cDNA library from senescent (24-month-old) rat liver mRNA and, by differential screening, have selected clones corresponding to mRNA species with increased abundance in aging rats. Direct sequencing of the inserts indicated that most of the clones (9 of 10) contained sequences coding for T-kininogen, also called major acute-phase protein, cysteine protease inhibitor, or thiostatin. Nuclear elongation experiments showed that the increase in mRNA concentration was controlled at the transcriptional level. RNase mapping and S1 analysis indicated that the age-dependent induction operated preferentially at one of the three transcriptional start sites of the gene(s). The acute-phase reaction (inflammation) is known to also induce these genes at the level of transcription; however, two of the three start sites are induced by inflammation. Transcription from one of these sites was induced by both phenomena, aging and inflammation.

Acute-Phase Proteins

Tissue-specific expression of beta-galactoside alpha-2,6-sialyltransferase. Transcript heterogeneity predicts a divergent polypeptide.

The beta-galactoside alpha-2,6-sialyltransferase represents a member of a family of sialyltransferases which catalyze the terminal addition of sialic acid to maturing carbohydrate chains. We surveyed rat tissues using cDNA probes complementary to coding and noncoding domains of the rat liver alpha-2,6-sialyltransferase. In addition to the expected differences in the level of sialyltransferase mRNA among the tissues, there were dramatic qualitative differences as well. Hepatic sialyltransferase probes hybridize to mRNAs of varying size on Northern blots. A tissue-dependent pattern of expression of these transcripts is documented. Evidence is presented that the multiple transcripts are generated from a common gene sequence. At least one instance of alternate splicing in the generation of the kidney sialyltransferase transcripts is predicted by S1 nuclease analysis. We report the isolation of a rat kidney cDNA clone, RKA, that substantiates this tissue-specific alternate splicing event. The RKA insert, although less than full-length, apparently encodes a polypeptide divergent from the reported hepatic alpha-2,6-sialyltransferase (1). RNA blot analysis indicates that the RKA-type transcripts represent a significant proportion of sialyltransferase RNA in rat kidney. Another class of kidney cDNA clones, RKE, is colinear with the hepatic sialyltransferase sequence. RNA blots probed for the divergent and common regions suggest that complex processing pathways are operative in the tissue-specific expression of sialyltransferase mRNA.

Amino Acid Sequence

Co-ordinate expression of cytochrome c oxidase subunit III and VIc mRNAs in rat tissues.

Cytochrome c oxidase (EC 1.9.3.1) is an enzyme which is composed of subunits derived from both the mitochondrial and the nuclear genomes. To determine whether or not the expression of these two genomes is co-ordinated at the mRNA level, we have examined the steady-state levels of mRNAs coding for cytochrome c oxidase subunit III (mitochondrially encoded) and subunit VIc (nuclear-encoded) in rat tissues. This was compared with the tissue concentration of the holoenzyme, which was estimated by measuring cytochrome c oxidase enzyme activity. The tissues (heart, brain, liver, kidney, soleus muscle and superficial white vastus muscle) possessed a 13-fold range of enzyme activity, which was highest in heart and lowest in the superficial vastus muscle. Specific subunit mRNA levels were quantified by using slot-blot hybridization of cDNA probes to total tissue RNA. The highest values for subunit III and Vlc mRNA tissue contents were found in kidney, followed by liver and heart (40-60% of that of kidney). The white vastus muscle contained the lowest subunit mRNA level (15% of that of kidney). Although some variability was apparent within each tissue, a parallel pattern of mRNA expression of the nuclear- and mitochondrially encoded subunits was observed. Differences between muscle (heart, vastus and soleus) and non-muscle tissues were noted in the relationship between mRNA and protein levels of expression. Thus, although this suggests that tissue-specific regulatory processes operate, the steady-state expression of subunit III and subunit Vlc mRNAs appears to be co-ordinately regulated.

Animals

Expression plasmid vectors containing Escherichia coli tryptophan promoter transcriptional units lacking the attenuator.

Two DNA fragments which contain the Escherichia coli tryptophan promoter operator region but lack the attenuator have been used in the construction of a series of pAT153 based plasmids suitable for the regulated expression of foreign genes in E. coli. The first, a 139-bp HhaI fragment includes 59 bp of the trp leader sequence, ending within the "attenuator peptide" coding sequence, eleven codons from the N-terminus. A fusion-type expression plasmid incorporating this fragment has been constructed. The second, a 99-bp HaeIII-TaqI fragment contains no coding sequence but includes the "attenuator peptide" SD site situated 4 bp upstream of the TaqI site. This fragment has been incorporated in expression vectors which result in the direct expression of cloned gene sequences. To further maximise expression, plasmids with directly repeating trp promoter HaeIII-TaqI units have been constructed.

Escherichia coli

A complex array of sequences enhances ribosomal transcription in Xenopus laevis.

The ribosomal DNA spacer in Xenopus laevis was shown in previous studies to be involved in regulating the expression of the ribosomal genes. Here transcription enhancement by this spacer has been studied in some detail, to fully identify the sequences involved and to determine their relative importance in this phenomenon. It is shown that the 60/81 base-pair (bp) repeats, which were reported to be enhancer elements, act as part of a mode of enhancement whose effect is amplified by the spacer promoters or Bam islands. The "Bam super repeat", a combination of spacer promoter and 60/81 bp elements, is the major enhancer unit. Within a Bam super repeat, a near linear correlation between the number of 60/81 bp elements and enhancer activity is observed. Thus, there is no significant co-operativity in the binding of transcription factors to an array of these elements. Multiple Bam super repeats do not act additively and may actually interfere with each others action. Surprisingly this effect is observed both in the presence and absence of active spacer promoters. Sequences between the 3' end of the 28 S coding region and the first spacer promoter may also be involved in enhancement but only in a very minor fashion. In confirmation of recent studies, the presence of the unique ribosomal termination sequence, 213 bp upstream from the pre-rRNA initiation site, is essential for efficient promotion, as deletion of this sequence virtually abolishes pre-rRNA- transcription. These data are discussed in terms of the possible mechanisms of transcription enhancement.

Animals

Prostaglandins and gene action: possible relevance to the effect of PG system on leukocyte alkaline phosphatase enzyme activity.

Prostaglandin E2, colchicine and imidazole, known to enhance and inhibit thromboxane A2 (TxA2) synthesis respectively and chloroquine, a PGE1 synthesis enhancer and PGE2 antagonist can alter the leukocyte alkaline phosphatase (LAP) enzyme activity. Thromboxane A2 and other related prostaglandins (PGs) are known to bind to DNA and thus possibly regulate DNA action. It is proposed here that prostaglandins are able to modify LAP activity by their action on the DNA and thus the Gene that codes for LAP. This hypothesis envisages that normally there are specific receptors on the genes for PGs. Taking LAP enzyme system as the model, it is proposed that the affinity of TxA1, TxA2, PGE2 and PGE1 to the operator is more than to that of the repressor. But when the levels of TxA2, PGE2 and PGE1 are in excess, all the receptors on the operator are not only completely occupied but they are also able to bind to repressor in sufficient amounts to transiently switch off the LAP synthesis by the structural gene. It is further envisaged that the affinity of the PG receptors on the operator and repressor systems of the operon is greatest for TxA1 and least for PGE2 (TxA1 greater than TxA2 greater than PGE1 greater than PGE2). This hypothesis though simply in its model explains all the variables obtained in our studies on the effect of PGs on the LAP activity. This concept by itself or a suitable modification, may explain the role of PGs in the pathogenesis of cancer. It will be interesting to study, in the light of this hypothesis, the role of the PG system on DNA repair mechanism, m-RNA and t-RNA synthesis and gene action in several systems.

Alkaline Phosphatase

Polymorphic sites and the mechanism of evolution in human mitochondrial DNA.

Twelve restriction enzymes were used to screen for the presence or absence of cleavage sites at 441 locations in the mitochondrial DNA of 112 humans from four continents. Cleavage maps were constructed by comparison of DNA fragment sizes with those expected from the published sequence for one human mtDNA. One hundred and sixty-three of the sites were polymorphic, i.e., present in some individuals but absent from others, 278 sites being invariant. These polymorphisms probably result from single base substitutions and occur in all functional regions of the genome.--In 77 cases, it was possible to specify the exact nature and location (within a restriction site) of the mutation responsible for the absence of a restriction site in a known human mtDNA sequence and its presence in another human mtDNA. Fifty-two of these 77 gain mutations occur in genes coding for proteins, 34 being silent and 18 causing amino acid replacements; moreover, nine of the replacements are radical.--Notable also is the anomalous ratio of transitions to transversions required to account for these 77 restriction site differences between the known human mtDNA sequences and other human mtDNAs. This ratio is lower for most groups of restriction sites than has been reported from sequence comparisons of limited parts of the mtDNA genome in closely related mammals, perhaps indicating a special functional role or sensitivity to mutagenesis for palindromic regions containing high levels of guanine and cytosine.--From the genomic distribution of the 163 polymorphic sites, it is inferred that the level of point mutational variability in tRNA and rRNA genes is nearly as high as in protein-coding genes but lower than in noncoding mtDNA. Thus, the functional constraints operating on components of the protein-synthetic apparatus may be lower for mitochondria than for other systems. Furthermore, the mitochondrial genes for tRNAs that recognize four codons are more variable than those recognizing only two codons.--Among the more variable of the human mitochondrial genes coding for proteins is that for subunit 2 of cytochrome oxidase; this polypeptide appears to have been evolving about five times faster in primates than in other mammals. Cytochrome c, a nuclearly encoded protein that interacts directly with the oxidase 2 subunit in electron transport, has also evolved faster in primates than in rodents or ungulates. This example, along with that for the mitochondrial rRNA genes and the nuclear genes coding for mitochondrial ribosomal proteins, provides evidence for coevolution between specific nuclear and mitochondrial genes.

Base Sequence

The yeast tRNA(Phe) gene family: structures and transcriptional activities reveal member differences not explained by intragenic promoters.

Several cloned members of the yeast tRNA(Phe) gene family were transcribed in vitro using a HeLa extract and a yeast extract. The optimum DNA concentration was determined and kinetic experiments were performed for each clone to compare transcription levels. Both extract systems were able to splice the intervening sequence, but only the yeast extract produced the mature product. Some genes were not transcribed with the homologous system while they were transcribed with the HeLa extract, suggesting a control mechanism that is not operating in the heterologous system. Competition experiments demonstrated that the intragenic promoters of the inactive genes were able to bind transcription factor(s), but not as efficiently as active genes. This binding was not so strong when using linear DNA and was dependent on the presence of the 3' intragenic control region. DNA sequencing and computer analysis indicated the presence of short conserved sequences upstream from the genes. These sequences, which are not related to the intragenic promoters, are direct repeats of part of the 3' coding region in those genes that are transcribed in the homologous system. The relevance of these sequences on homologous transcription in vitro remains to be established.

Base Sequence

Structure and regulated expression of the SpoC1 gene cluster from Aspergillus nidulans.

We have previously described the organization of a 13.3 kb region of the Aspergillus nidulans genome, designated SpoC1, coding for multiple poly(A)+ RNAs that accumulate in asexual spores but not in somatic cells. We have determined the limits of the SpoC1 gene cluster by investigating the transcriptional features of 53 kb of chromosomal DNA. This segment of the genome codes for at least 19 poly(A)+ RNAs, some of which are transcribed from overlapping regions. The area of developmental regulation is approximately 38 kb in length and is delimited by 1.1-kb direct repeats. With one exception, RNAs transcribed from the central part of the cluster appear late during conidiophore development and accumulate specifically in spores. The exceptional transcript appears earlier during development and accumulates specifically in cells of the conidiophore. In contrast, RNAs encoded at the borders of the cluster occur in both somatic cells and spores. The results indicate that if a chromatin-level control mechanism operates to regulate expression of the SpoC1 gene cluster, as previously suggested by us, additional levels of regulation must also exist.

Aspergillus nidulans

Transcriptional activation and stabilization of malic enzyme mRNA precursor by thyroid hormone.

One of the responses to the administration of thyroid hormone is an increase in malic enzyme (EC 1.1.1.40) mRNA in rat liver. We have previously shown that 3,5,3'-triiodo-L-thyronine (T3) causes a 3-4-fold increase in the rate of transcription of the malic enzyme gene as determined by in vitro run-off assays with the cDNA probe following T3 treatment for 10 days (Dozin, B., Magnuson, M.A., and Nikodem, V. M. (1986) J. Biol. Chem. 261, 10290-10292). Since the level of cytoplasmic mRNA increases 10-15-fold, one or more additional mechanisms must be operative to produce the full effect. We have now analyzed the time course of the effect of T3 on the rate of transcription and the accumulation of malic enzyme RNA in the nucleus using malic enzyme cDNAs and intronic probes. There is an approximately 10-12-fold increase in the level of nuclear RNA accompanied by the same increase in cytoplasmic mRNA, showing a half-rise time of about 60 h. The 3-4-fold increase in the transcription rate occurred with a half-time of about 18 h. The relative values for either the increase in transcriptional activity or the increase in the level of malic enzyme RNA in the nucleus were identical irrespective of the probes used. As a control, we examined the effect of a high carbohydrate diet which is known to increase malic enzyme mRNA without affecting either transcriptional rate or nuclear RNA (Dozin, B., Rall, J. E., and Nikodem, V. M. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 4705-4709). As expected, no change in the level of malic enzyme RNA in the nucleus was found with the intronic probes. We conclude that T3 both activates transcription of the malic enzyme gene in rat liver and decreases the rate of degradation of pre-mRNA coding for malic enzyme.

Animals

Arginine repression of the Saccharomyces cerevisiae ARG1 gene. Comparison of the ARG1 and ARG3 control regions.

The Saccharomyces cerevisiae ARG1 gene coding for argininosuccinate synthetase has been isolated and the nucleotide sequence of both its control region and of its amino terminal end coding region determined. The startpoint of transcription was established by S1-mapping and reverse transcriptase procedures. Northern blot hybridizations showed that whereas arginine-specific repression reduced the enzyme activity fivefold, it did not reduce the steady state level of the corresponding messenger in proportion; by analogy with the coregulated ARG3 gene, this result suggests a post-transcriptional regulatory mechanism. In contrast, proportionally between enzyme activity and mRNA content was observed under conditions where general amino acid control (known to be transcriptional) was operating. Comparing the 5' untranscribed domains of ARG1 and ARG3 revealed a first region of homology between the TATA box and the transcription startpoint. In this region a 10 bp (ARG3) or 11 bp (ARG1) central box is flanked by two segments which, by mutation, have been shown to be part of the ARG operator (Crabeel et al. 1985). The repressor is assumed to bind at this primary target site prior to establishing contacts with the proximal part of the nascent mRNA molecule (Crabeel et al. 1985). By in vitro directed deletion mutagenesis we show that the central conserved box of ARG3 is not essential for arginine-specific repression to occur. Another region of homology was found in the leader part of the messenger RNA; deletion of this region causes a small reduction in ARG3 expression but also does not alter regulation. Neither of these two regions are thus part of the primary repressor target site. In addition, in terms of post-transcriptional regulation, the latter result indicates that no sequence specificity is required in the RNA recognition step.

Amino Acid Sequence

Structure and regulation of the glpFK operon encoding glycerol diffusion facilitator and glycerol kinase of Escherichia coli K-12.

The glpFK operon maps near minute 88 on the linkage map of Escherichia coli K-12 with glpF promoter proximal. The glpF gene encodes a cytoplasmic membrane protein which facilitates the diffusion of glycerol into the cell. The glpK gene encodes glycerol kinase. In the present work, the nucleotide sequence of the 5'-end of the operon, including the control region, the glpF gene, and part of the glpK gene, was determined. The facilitator was predicted to contain 281 amino acids with a calculated molecular weight of 29,780. It is a highly hydrophobic protein with a minimum of six potential transmembrane alpha helices. The transcription start site for the glpFK operon was located 71 base pairs upstream from the proposed translation start codon for glpF. Preceding the transcription start site were sequences similar to the -10 and -35 consensus sequences for bacterial promoters. Binding sites for the cAMP-cAMP receptor protein (CRP) complex and the glp repressor were identified by DNase I footprinting. The region protected by the cAMP.CRP complex contained tandem sequences resembling the consensus sequence for CRP binding. The CRP sites were centered at 37.5 and 60.5 base pairs upstream of the start of transcription. The glp repressor protected an extensive area (-89 to -7 relative to the start point of transcription), sufficient for the binding of four repressor tetramers. Two additional binding sites for the repressor were identified within the glpK coding region. The DNA containing these two operators synergistically increased the apparent affinity of glp repressor for DNA fragments containing the four operators in the promoter region of the glpFK operon. With this study, a total of 13 operators for the glp regulon have been characterized. Comparison of these operators revealed the consensus 5'-WATGTTCGWT-3' for the operator half-site (W = A or T). The relative affinity of the glp repressor for the various glp operators was assessed in vivo using a promoter-probe vector. The relative apparent affinity of the control regions for glp repressor was glpFK greater than glpD greater than glpACB greater than glpTQ. The degree of catabolite repression for each of the operons was assessed using a similar system. In this case, the relative sensitivity of the glp operons to catabolite repression was glpTQ greater than glpFK greater than glpACB greater than glpD.

Amino Acid Sequence

Analysis of alternatively spliced human immunodeficiency virus type-1 mRNA species, one of which encodes a novel tat-env fusion protein.

A polymerase chain reaction-based analysis was used to define the structures of the mRNAs that encode human immunodeficiency virus type-1 (HIV-1) regulatory and structural proteins in infected H9 cells. Twenty alternatively spliced mRNAs encoding the vif, vpr, env, nef, tat, and rev proteins were characterized. An evaluation of the coding potentials of these transcripts recognized both leaky scanning and reinitiation at downstream initiation codons as mechanisms that may operate during translation of many of the polycistronic messages. Two new splice acceptor sites, one at nt 6018 defining a new mRNA coding for the env and vpu proteins and another at nt 8671 defining a novel tat-env fusion transcript, were characterized. The latter transcript expressed a novel protein p17tev that was immunoprecipitated by both polyclonal tat antibodies and monoclonals directed towards the C-terminal region of gp41. The p17tev protein was able to transactivate transcription from the HIV-1 LTR in transient transfection assays. The use of multiple alternative splice donor and acceptor sites and the generation of novel proteins may confer evolutionary advantages on the viral mutants encoding them and influence the course of clinical disease.

Animals

Alternative splicing dictates translational start in Epstein-Barr virus transcripts.

Of the 10 viral genes known to be expressed during Epstein-Barr virus (EBV) latency, six encode nuclear antigens (EBNAs), all of which are expressed from a long primary transcript by means of alternative splicing and alternative polyadenylation sites. The primary transcript is generated by either of two promoters which operate in a mutually exclusive fashion in different clonal cell lines. All mRNAs from either promoter have exons in common from the BamHI W viral genomic fragment (the major internal repeat, IR1) which encode the N-terminal portion of one of the nuclear antigens (EBNA 4). In addition to the coding regions for EBNA 4, EBNA mRNAs encode another EBNA (i.e. EBNA 1, 2, 3A, 3B or 3C) downstream. We show that alternative splicing determines whether the translation initiation codon for EBNA 4 is present or absent, thus permitting the generation of mRNAs in which the first translation initiation codon is either that for the EBNA 4 gene or for the other EBNA gene encoded downstream. This mechanism presumably ensures efficient translation of all the EBNA genes.

Amino Acid Sequence

Assay systems for the study of gene function. 2 assay systems, the syntheses of RNA and protein, are described; their virtues and drawbacks are discussed.

The study of genetic regulatory mechanisms operating in plants and animals is of paramount importance in contemporary biology. A precise understanding of the mechanisms that underlie normal cellular differentiation is a prerequisite for understanding neoplastic transformation and genetic disease. At present, we are not aware of a single assay system that can give answers to all questions we are already able to pose. Studies of RNA synthesis are valuable because they provide a direct measurement of transcriptional activity. But these studies remain incomplete until we succeed in unraveling the metabolic roles of the molecules whose synthesis we study. In this respect, the study of enzyme synthesis represents a better defined assay system, although the interpretation of observed fluctuations in synthetic rates is made difficult by the many steps that intervene between the genes and their finished protein products. We propose that a combination of protein biosynthetic and cytogenetic analysis is a promising assay system for further investigation.

Alcohol Oxidoreductases

Messenger RNAs coding for receptors and channels in the cerebral cortex of adult and aged rats.

Poly(A)+ mRNAs from the cerebral cortex of aged (24 months) and young adult (3 months) rats were isolated and injected into Xenopus oocytes to express functional neurotransmitter receptors and voltage-operated channels. Electrophysiological recordings of induced membrane currents were used as a measure of the relative amounts of mRNA encoding different receptors and channels, and to study their functional properties. There were no large differences apparent between mRNAs from aged and adult rats, in marked contrast to the dramatic (1000-fold) changes in mRNA expression that occur during embryonic and postnatal development. The membrane currents induced by glutamate or acetylcholine (ACh) application were roughly one third smaller in oocytes injected with mRNA from aged cerebral cortex than in oocytes injected with mRNA from adult cerebral cortex, whereas currents induced by gamma-aminobutyric acid (GABA), kainate or serotonin (5-HT) application, and by activation of voltage-operated Na+ and Ca2+ channels were not significantly different. We did not observe any age-related differences in the properties of the receptors and channels studied.

Acetylcholine

cDNA cloning and functional expression in yeast Saccharomyces cerevisiae of beta-naphthoflavone-induced rabbit liver P-450 LM4 and LM6.

A cDNA library was constructed from liver mRNA of a beta-naphthoflavone-induced rabbit. Two clones pLM4-1 and pLM6-1 containing 2.2-kbp inserts that hybridized at low stringincy with a mouse P1 P-450 probe were selected. The clone pLM4-1 was fully sequenced and found to contain a full-length cDNA coding for cytochrome P-450 LM4. Partial sequence and restriction mapping made it possible to identify pLM6-1 as coding for the major part of cytochrome P-450 LM6. Cloned LM4-1 cDNA was reformed by deletion of the 5' and 3' non-coding regions before insertion into yeast expression vectors PYe DP1/10. A similar operation was performed on pLM6-1 cDNA after replacement of the missing N-terminus-coding sequences by homologous sequences form the pLM4-1 clone resulting in a chimeric cytochrome P-450 coding sequence. Expression of cloned rabbit cytochrome P-450 into transformed yeast was optimized by studying the effect of the nature of the DNA sequence just preceding the initiation codon on the level of cytochrome P-450 production. Yeast synthesized cytochromes P-450 were characterized by immunoblotting, spectra and catalytic activity determinations. Cloned cytochrome P-450 LM4 was found by all criteria to be identical to the authentic rabbit one. The chimeric cytochrome P-450 that contains the 143 N-terminal amino acids of cytochrome P-450 LM4 and the remaining 375 amino acids of cytochrome P-450 LM6 was found to exhibit most of the authentic cytochrome P-450 LM6 catalytic properties. Enzymatic and evolutionary implications of these results are discussed.

Amino Acid Sequence

Molecular analysis of the endosymbionts of tsetse flies: 16S rDNA locus and over-expression of a chaperonin.

Based on 16S rDNA sequence comparison, intracellular mycetome-associated endosymbionts (P-endosymbionts) of tsetse flies (Diptera: Glossinidae) form a distinct lineage within the gamma-3 subdivision of proteobacteria, related to the free-living bacterium Escherichia coli, midgut S-endosymbionts of various insects including tsetse flies, and to the P-endosymbiont lineage of aphids, Buchnera aphidicola. Gene organization and expression of several loci in intracellular microorganisms have revealed differences from free-living bacteria. This study analyses two of these characteristics in tsetse endosymbionts; the copy number and gene organization of rDNA operations and the nature of the abundant protein(s) synthesized by these microorganisms. Results indicate that Glossina morsitans morsitans S-endosymbionts have multiple (seven) rDNA operons coding for 16S (rrs) followed by 23S (rrl) gene sequences, whereas tsetse P-endosymbionts have a single, similarly organized rDNA operon. In tsetse mycetocytes in vitro, P-endosymbionts synthesize a predominant protein of 60 kDa in size (p60) which by Western blot analysis shows immunological cross-reactivity with the abundant 63 kDa (p63) protein of B. aphidicola. p63 (also referred to as symbionin) has been characterized as a molecular chaperone, structurally and functionally similar to the groEL protein of E. coli. Under in vitro conditions, tsetse S-endosymbionts synthesize high levels of a similarly-sized protein that cross-reacts with p63 chaperonin. Antisera against the tsetse p60 protein also recognizes p63 protein of B. aphidicola, suggesting that the abundant tsetse endosymbiont protein is a chaperonin.

Animals