Purification of mRNA coding for rat-liver fructose-1,6-bisphosphatase by polysome immunoabsorption.

The mRNA coding for rat liver fructose-1,6-bisphosphatase, which represents approx. 0.46% of total hepatic mRNA, has been purified to near homogeneity. Polysomes from rat liver were allowed to react with antibodies to rabbit anti-fructose-1,6-bisphosphatase purified by affinity chromatography. The complex was immobilized on a protein A-Sepharose column. After the removal of unabsorbed polysomes, the specific mRNA was eluted and chromatographed on an oligo(dT)-cellulose column. This method gave a 183-fold enrichment of the fructose-1,6-bisphosphatase mRNA to greater than 80% homogeneity as determined by electrophoreses of immunoprecipitated in vitro translation products on polyacrylamide slab gels in the presence of sodium dodecyl sulphate.

Purification and properties of rabbit heart muscle aldolase.

Fructose diphosphate aldolase (D-fructose-1,6-biphosphate D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13) from rabbit heart has been purified and obtained in crystalline form. The preparations are homogeneous on the basis of disc gel electrophoresis and ultracentrifugation. The catalytic and the molecular properties indicate that this is aldolase A. A comparison was made between rabbit heart aldolase and the rabbit muscle enzyme. The sedimentation coefficient, energy of activation and Michaelis constant for Fru-1,6-P2 were found to be identical with the values obtained for the muscle enzyme. As in case of the muscle enzyme, heart aldolase was found to have a broad pH optimum, remarkable stability over a wide pH range, and the ability to form a Schiff base intermediate with dihydroxyacetone phosphate upon reduction with borohydride. Cleavage of the methionyl bonds with CNBr yields the same pattern as obtained with the muscle enzyme.

Studies on the structure of aldolase A from chicken muscle.

S-Carboxymethylated chicken muscle aldolase was treated with cyanogen bromide to cleave the 4 methionyl bonds per subunit. Five homogeneous fractions were obtained designated fragments I-V. Fragment I was derived from the N-terminus and fragment II from the C-terminus of the enzyme. Reduction of the enzyme with NaB3H4 in the presence of dihydroxyacetone phosphate decreases the enzymatic activity by 90%. Fragment III contained the Schiff base-forming lysine residue since more than 83% of the radioactivity introduced by NaB3H4 reduction of aldolase-dihydroxyacetone phosphate was found in this fraction. A tryptic peptide of 27 amino acid residues containing the substrate-binding site was isolated. The gross molecular structure of aldolase A from chicken muscle indicates a high degree of homology with mammalian muscle aldolases.

Mutants of Trichoderma reesei are defective in cellulose induction, but not basal expression of cellulase-encoding genes.

Four mutants of Trichoderma reesei defective in cellulose utilization were characterized at the molecular level. Genomic analysis of the cellulase-encoding genes (cel) and transcript induction using two well-established inducers of the cel system--the insoluble polymer, cellulose and the soluble inducer, sophorose,--revealed that these mutants are defective in the transcription of cel genes. The results also indicate that the cel genes are coordinately expressed and most probably are regulated by the same mechanism. Using a heterologous gene construct, in which the hygromycin-B-resistance-encoding gene was placed under the control of the promoter of the major cel gene, cbh1, we showed that the mutants synthesize basic levels of cellulase, but are defective in the cel induction.

Structure, organization and promoter expression of the actin-encoding gene in Trichoderma reesei.

The single gene encoding actin (Act) in the cellulolytic filamentous fungus Trichoderma reesei (Tr) has been isolated and characterized. The gene contains five introns located in identical positions when compared to the putative ancestral actin genes (act) present in Thermomyces lanuginosus and Aspergillus nidulans. The 5' untranslated region (UTR) of the gene contains a TATA-like sequence (TAATA), a C + T-rich region and a potential CCAAT motif. This region was used as a homologous promoter to direct expression of hygromycin-B-resistance-encoding gene as a dominant-selectable Tr marker.

Transcriptional control of the cellulase genes in Trichoderma reesei.

The expression of the cellulase transcripts of Trichoderma reesei is controlled by the nature of the energy carbon sources used in the culture medium. Cellulose and the soluble disaccharide sophorose, but not glycerol or glucose, act as inducers. Evidence is presented suggesting that a low constitutive extracellular cellulolytic system catalyzes the formation of a soluble inducer from cellulose, and this inducer triggers the expression of the cellulase transcripts. This basal and cellulose-induced expression of the cellobiohydrolase I mRNAs (cbh1), the major member of the cellulase system, is transcriptionally controlled by two independent cis-acting DNA regions. In addition, expression of the cbh1 transcript is influenced by the physiological state of the mitochondria and this sensitivity is controlled through the 5'-flanking DNA sequence of this gene.

Angiotensin-converting enzyme: immunologic, structural, and developmental aspects.

Immunization of dog and rat high pure rabbit pulmonary angiotensin-converting enzyme elicited, in some individuals, antibodies that inhibited their own converting enzyme. Active immunization with an immunologically related enzyme is thus a plausible approach for developing biologically based inhibitors of enzymes that are either in or accessible to the circulation. Rabbit testicular peptidyldipeptide hydrolase was purified to homogeneity and found to be a considerably smaller (Mr approximately 100,000) glycoprotein than pulmonary converting enzyme (Mr approximately 140,000). The two enzymes differed at their amino- and carboxy-termini. However, they exhibited identical catalytic properties, and antibodies prepared against either inhibited both similarly. In competition radioimmunoassays, antibodies against the pulmonary enzyme preferred it to the testicular species, whereas those against the latter did not distinguish between the two molecules. The testicular isozyme thus resembles an internal part of the pulmonary polypeptide, which includes its active site. In a reticulocyte lysate, mRNA from the lungs of immature and mature rabbits comparably primed the synthesis of a polypeptide (Mr approximately 129,000) that reacted with anticonverting enzyme antibodies. In contrast, an immunoreactive species was programed only by mRNA from the testis of mature animals, and this protein was much smaller (Mr approximately 85,000). Maturation dependence and a shorter polypeptide chain, the regulatory and structural properties that distinguish the testicular isozyme, are thus each pretranslationally determined.

Phosphofructokinase from bumblebee flight muscle. Molecular and catalytic properties and role of the enzyme in regulation of the fructose 6-phosphate/fructose 1,6-bisphosphate cycle.

Phosphofructokinase from the flight muscle of bumblebee was purified to homogeneity and its molecular and catalytic properties are presented. The kinetic behavior studies at pH 8.0 are consistent with random or compulsory-order ternary complex. At pH 7.4 the enzyme displays regulatory behavior with respect to both substrates, cooperativity toward fructose 6-phosphate, and inhibition by high concentration of ATP. Determinations of glycolytic intermediates in the flight muscle of insects exposed to low and normal temperatures showed statistically significant increases in the concentrations of AMP, fructose 2,6-bisphosphate, and glucose 6-phosphate during flight at 25 degrees C or rest at 5 degrees C. Measuring the activity of phosphofructokinase and fructose 1,6-bisphosphatase at 25 and 7.5 degrees C, in the presence of physiological concentrations of substrates and key effectors found in the muscle of bumblebee kept under different environmental temperatures and activity levels, suggests that the temperature dependence of fructose 6-phosphate/fructose 1,6-bisphosphate cycling may be regulated by fluctuation of fructose 2,6-bisphosphate concentration and changes in the affinity of both enzymes for substrates and effectors. Moreover, in the presence of in vivo concentrations of substrates, phosphofructokinase is inactive in the absence of fructose 2,6-bisphosphate.

Mechanism by which cellulose triggers cellobiohydrolase I gene expression in Trichoderma reesei.

The expression of cellobiohydrolase I mRNA from Trichoderma reesei, measured by Northern blot hybridization, is controlled by the nature of carbon sources used in the culture medium. Cellulose and the soluble disaccharide sophorose, but not glycerol or glucose, act as inducers. Cellobiohydrolase I mRNA was undetectable when antibodies to the major members of the cellulolytic system were present in the culture medium prior to the addition of cellulose. These antibodies had no repressive effect if sophorose was used as an inducer. The results strongly suggest that a low constitutive cellulolytic system catalyzes the formation of a soluble inducer from cellulose and that this inducer triggers the expression of the cellobiohydrolase I gene transcript, most probably at the transcription level.

Evidence for nuclear targeting of prothymosin and parathymosin synthesized in situ.

To test the hypothesis that prothymosin and parathymosin contain amino acid sequences that cause them to be targeted to the cell nucleus, expression vectors were constructed containing a simian virus 40 promoter and cDNAs that would code for chimeric proteins composed of truncated human growth hormone (hGH) linked to the NH2 terminus of prothymosin or parathymosin. The truncated hGH lacked the signal peptide sequence required for its secretion. After transfection of these constructs into HeLa S3 cells, which do not normally synthesize hGH, the use of indirect immunofluorescence staining to follow the localization of the hGH chimeras demonstrated that both prothymosin and parathymosin caused targeting to the cell nucleus. Controls with a construct coding for native hGH only, and one coding for the truncated hGH lacking the signal peptide, revealed secretion into culture medium and staining in the endoplasmic reticulum and Golgi apparatus in the first case, and diffuse staining throughout the cytoplasm in the second. The results provide direct evidence, with proteins synthesized in situ, for the presence of nuclear localization signals in both prothymosin and parathymosin.

Dipeptidyl carboxypeptidase from seminal fluid resembles the pulmonary rather than the testicular isoenzyme.

The specific activity, molecular weight and immunological behavior of pure dipeptidyl carboxypeptidase from rabbit seminal fluid were found to resemble the corresponding properties of the pulmonary rather than the testicular isozyme.

Tissue-specific expression of mRNAs for dipeptidyl carboxypeptidase isoenzymes.

The molecular weight of newly synthesized dipeptidyl carboxypeptidase (angiotensin-converting enzyme; peptidyldipeptide hydrolase, EC 3.4.15.1) polypeptide primed in a reticulocyte lysate by poly(A)-containing RNA from mature rabbit testis was only about 65% that of the immunologically related species programmed by pulmonary RNA. Furthermore, in contrast to the pulmonary RNA-dependent product, the synthesis of this testicular protein was not directed by RNA from testes of immature animals. These findings indicate that a shorter polypeptide chain and pubertal expression--the structural and regulatory properties that distinguish the testicular dipeptidyl carboxypeptidase isozyme--are determined pretranslationally.

Two regulatory regions controlling basal and cellulose-induced expression of the gene encoding cellobiohydrolase I of Trichoderma reesei are adjacent to its TATA box.

The cellulolytic system of the filamentous fungus Trichoderma reesei is transcriptionally induced in the presence of the insoluble polymer cellulose. Previous studies have demonstrated that induction of the cellulose transcripts by cellulose requires basal expression of its own genes. To understand how basal expression controls cellulose-induced transcription of those genes, we analyzed the 5'-flanking region of the gene encoding cellobiohydrolase I (cbh1), the major member of the cellulase system, for the cis-acting region that is responsible for regulating basal and cellulose-stimulated expression. Using the promoter deletion approach and an appropriate reporter gene, the cis-acting region responsible for cellulose-stimulated transcription was localized between -241 and -72 bp relative to the TATA box. Deletion of this sequence did not affect the basal expression of the promoter, whereas deletion of 72 bp adjacent to the TATA box abolished basal expression of the cbh1 promoter. We therefore concluded that the cbh1 promoter is composed of two regulatory regions-one controls cellulose-induced transcription and the other is required for its basal expression.

Mitochondrial functions mediate cellulase gene expression in Trichoderma reesei.

We examined the effects of inhibition of mitochondrial functions on the expression of two nuclear genes encoding the extracellular cellobiohydrolase I (cbh1) and endoglucanase I (egl1) of the cellulase system of the filamentous fungus Trichoderma reesei. The cbh1 and egl1 transcripts are repressed at a low oxygen tension, and by glucose at a concentration known to repress mitochondrial respiration. The transcripts are also down-regulated by chemical agents known to dissipate the proton electrochemical gradient of the inner mitochondrial membrane and blocking of the electron-transport chain, such as DNP and KCN, respectively. These results suggest that expression of those transcripts is influenced by the physiological state of the mitochondria. In addition, heterologous gene fusion shows that the sensitivity of the expression of those transcripts to the functional state of the mitochondria is transcriptionally controlled through the 5'-flanking DNA sequence of those genes.

Molecular and catalytic properties of rabbit testicular dipeptidyl carboxypeptidase.

Rabbit testicular dipeptidyl carboxypeptidase activity was purified by a procedure exploiting its affinity for N-alpha-[1-(S)-carboxy-3-phenylpropyl]-L-lysyl-L-proline. The molecular, catalytic, and immunological properties of the testicular enzyme are presented and compared with the corresponding properties of pulmonary angiotensin-converting enzyme. Although catalytically similar and immunologically related to pulmonary dipeptidyl carboxypeptidase, the testicular enzyme has a molecular weight (100,000) which is lower by a factor of about one-third and differs in its NH2 and COOH termini. Furthermore, we present evidence that the testicular enzyme is not a post-translation product of the pulmonary type enzyme. These data suggest that testicular and pulmonary dipeptidyl carboxypeptidase are two distinct proteins which are catalytically similar and immunologically closely related.

A region of the cellobiohydrolase I promoter from the filamentous fungus Trichoderma reesei mediates glucose repression in Saccharomyces cerevisiae, dependent on mitochondrial activity.

The upstream activating region that controls cellulose-induced expression of the glucose-repressible cellobiohydrolase I gene (UARcb1) of the filamentous fungus Trichoderma reesei is shown to mediate transcription and glucose repression of a reporter gene in Saccharomyces cerevisiae, a unicellular microorganism that lacks the genes required for the utilization of cellulose. Glucose-controlled transcription mediated by UARcb1 requires the products of the genes SNF1 and SSN6, a protein kinase and a repressor, respectively, that regulate glucose-repressible yeast genes. Previously, it has been shown that mitochondrial function is implicated in cellobiohydrolase I gene expression in T. reesei and this sensitivity to the metabolic state of the mitochondria was shown to be transcriptionally controlled by the 5'-flanking sequence of the cbh1 gene [Abrahão-Neto et al. (1995) Biochemistry 34, 10456-10462]. Remarkably, transcription of the reporter gene controlled by UARcb1 in S. cerevisiae also showed a requirement for active mitochondria, suggesting that a common mechanism involving mitochondrial activity controls glucose-repressible genes in both microorganisms.

Cellulase induction in Trichoderma reesei by cellulose requires its own basal expression.

The induction of cellulases by cellulose, an insoluble polymer, in the filamentous fungus Trichoderma reesei is puzzling. We previously proposed a mechanism that is based on the presence of low levels of cellulase in the uninduced fungus; this basal cellulase activity would digest cellulose-releasing oligosaccharides that could enter the cell and trigger expression of cellulases. We now present experiments that lend further support to this model. We show here that transcripts of two members of the cellulase system, cbh1 and egl1, are present in uninduced T. reesei cells. These transcripts are induced at least 1100-fold in the presence of cellulose. We also show that a construct containing the hygromycin B resistance-encoding gene driven by the cbh1 promoter confers hygromycin B resistance to T. reesei cells grown in the absence of cellulose. Moreover, cellulose-induced production of the cbh1 transcript was suppressed when antisense RNA against three members of the cellulase system was expressed in vivo. Experiments are presented indicating that extracellular cellulase activity is the rate-limiting event in induction of synthesis of the cellulase transcripts by cellulose. The results reveal a critical requirement for basal expression of the cellulase system for induction of synthesis of its own transcripts by cellulose.

Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags.

Transcribed sequences in the human genome can be identified with confidence only by alignment with sequences derived from cDNAs synthesized from naturally occurring mRNAs. We constructed a set of 250,000 cDNAs that represent partial expressed gene sequences and that are biased toward the central coding regions of the resulting transcripts. They are termed ORF expressed sequence tags (ORESTES). The 250,000 ORESTES were assembled into 81,429 contigs. Of these, 1, 181 (1.45%) were found to match sequences in chromosome 22 with at least one ORESTES contig for 162 (65.6%) of the 247 known genes, for 67 (44.6%) of the 150 related genes, and for 45 of the 148 (30.4%) EST-predicted genes on this chromosome. Using a set of stringent criteria to validate our sequences, we identified a further 219 previously unannotated transcribed sequences on chromosome 22. Of these, 171 were in fact also defined by EST or full length cDNA sequences available in GenBank but not utilized in the initial annotation of the first human chromosome sequence. Thus despite representing less than 15% of all expressed human sequences in the public databases at the time of the present analysis, ORESTES sequences defined 48 transcribed sequences on chromosome 22 not defined by other sequences. All of the transcribed sequences defined by ORESTES coincided with DNA regions predicted as encoding exons by genscan. (http://genes.mit.edu/GENSCAN.html).

The genome sequence of the plant pathogen Xylella fastidiosa. The Xylella fastidiosa Consortium of the Organization for Nucleotide Sequencing and Analysis.

Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes a range of economically important plant diseases. Here we report the complete genome sequence of X. fastidiosa clone 9a5c, which causes citrus variegated chlorosis--a serious disease of orange trees. The genome comprises a 52.7% GC-rich 2,679,305-base-pair (bp) circular chromosome and two plasmids of 51,158 bp and 1,285 bp. We can assign putative functions to 47% of the 2,904 predicted coding regions. Efficient metabolic functions are predicted, with sugars as the principal energy and carbon source, supporting existence in the nutrient-poor xylem sap. The mechanisms associated with pathogenicity and virulence involve toxins, antibiotics and ion sequestration systems, as well as bacterium-bacterium and bacterium-host interactions mediated by a range of proteins. Orthologues of some of these proteins have only been identified in animal and human pathogens; their presence in X. fastidiosa indicates that the molecular basis for bacterial pathogenicity is both conserved and independent of host. At least 83 genes are bacteriophage-derived and include virulence-associated genes from other bacteria, providing direct evidence of phage-mediated horizontal gene transfer.

Comparative genomics of two Leptospira interrogans serovars reveals novel insights into physiology and pathogenesis.

Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in accidental hosts, including humans. Complete genome sequencing of Leptospira interrogans serovar Copenhageni and comparative analysis with the available Leptospira interrogans serovar Lai genome reveal that despite overall genetic similarity there are significant structural differences, including a large chromosomal inversion and extensive variation in the number and distribution of insertion sequence elements. Genome sequence analysis elucidates many of the novel aspects of leptospiral physiology relating to energy metabolism, oxygen tolerance, two-component signal transduction systems, and mechanisms of pathogenesis. A broad array of transcriptional regulation proteins and two new families of afimbrial adhesins which contribute to host tissue colonization in the early steps of infection were identified. Differences in genes involved in the biosynthesis of lipopolysaccharide O side chains between the Copenhageni and Lai serovars were identified, offering an important starting point for the elucidation of the organism's complex polysaccharide surface antigens. Differences in adhesins and in lipopolysaccharide might be associated with the adaptation of serovars Copenhageni and Lai to different animal hosts. Hundreds of genes encoding surface-exposed lipoproteins and transmembrane outer membrane proteins were identified as candidates for development of vaccines for the prevention of leptospirosis.