Variations and coding features of the sequence spanning the replication terminus of Bacillus subtilis 168 and W23 chromosomes.

In a comparative study of the sequences of the 3-kb regions of DNA spanning the replication terminus, terC, of Bacillus subtilis strains 168 and W23, it was found that the latter contained an insertion of a large open reading frame (ORF405) whose translated protein product is a member of the cytochrome P-450 family. The insertion was about 34 nucleotides upstream from a putative promoter for the rtp gene. The sequenced regions contained a number of other ORFs. The translation product of one (ORF238) is a member of a previously identified oxidoreductase superfamily. The translation product of another (ORF257) is significantly similar to the proC product of Escherichia coli, but this ORF does not code for a functional proC product of B. subtilis.     

Structure and deduced function of the granaticin-producing polyketide synthase gene cluster of Streptomyces violaceoruber Tü22.

A 6.5 kb region of DNA from Streptomyces violaceoruber, which contains polyketide synthase (PKS) genes for production of the benzoisochromane quinone moiety of the antibiotic, granaticin, was cloned and sequenced. Of six open reading frames (ORFs) identified, four (ORFs 1-4) would be transcribed in one direction and two (ORFs 5 and 6) divergently from ORFs 1-4. ORF1 and ORF2, which show evidence for translation coupling, encode (deduced) gene products which strongly resemble each other and the Escherichia coli fatty acid ketoacyl synthase (condensing enzyme), FabB. We conclude that ORF1 (which contains a characteristic cysteine residue) functions as a condensing enzyme, possibly as part of a heterodimeric protein including the product of ORF2. The predicted ORF3 gene product strikingly resembles acyl carrier proteins (ACPs) of fatty acid synthase (FAS), particularly in the region of the active site motif, while the predicted ORF5 and ORF6 gene products resemble known oxidoreductases, suggesting that they function as reductive steps required during assembly of the granaticin carbon skeleton. Comparison of the deduced ORF4 gene product with available protein databases failed to elucidate its potential function. The overall conclusion is that the granaticin-producing PKS would consist of at least six separate enzymes involved in carbon chain assembly, thus resembling a Type II, rather than a Type I, FAS.     

Time-dependent increase in ribosome processivity

We created a novel tripartite reporter RNA to separately and simultaneously examine ribosome translation rates at the 5′- and 3′-ends of a large open reading frame (ORF) in vitro in HeLa cell lysates. The construct contained Renilla luciferase (RLuc), β-galactosidase and firefly luciferase (FLuc) ORFs linked in frame and separated by a viral peptide sequence that causes cotranslational scission of emerging peptide chains. The length of the ORF contributed to low ribosome processivity, a low number of initiating ribosomes completing translation of the entire ORF. We observed a time-dependent increase in FLuc production rate that was dependent on a poly(A) tail and poly(A)-binding protein, but was independent of eIF4F function. Stimulation of FLuc production occurred earlier on shorter RNA templates. Cleavage of eIF4G at times after ribosome loading on templates occurred did not cause immediate cessation of 5′-RLuc translation; rather, a delay was observed that shortened when shorter templates were translated. Electron microscopic analysis of polysome structures in translation lysates revealed a time-dependent increase in ribosome packing and contact that correlated with increased processivity on the FLuc ORF. The results suggest that ORF transit combined with PABP function contribute to interactions between ribosomes that increase or sustain processivity on long ORFs.     

Steroid hormone regulation of the Achlya ambisexualis 85-kilodalton heat shock protein, a component of the Achlya steroid receptor complex.

The steroid hormone antheridiol regulates sexual development in the fungus Achlya ambisexualis. Analyses of in vivo-labeled proteins from hormone-treated cells revealed that one of the characteristic antheridiol-induced proteins appeared to be very similar to the Achyla 85-kilodalton (kDa) heat shock protein. Analysis of in vitro translation products of RNA isolated from control, heat-shocked, or hormone-treated cells demonstrated an increased accumulation of mRNA encoding a similar 85-kDa protein in both the heat-shocked and hormone-treated cells. Northern (RNA) blot analyses with a Drosophila melanogaster hsp83 probe indicated that a mRNA species of approximately 2.8 kilobases was substantially enriched in both heat-shocked and hormone-treated cells. The monoclonal antibody AC88, which recognizes the non-hormone-binding component of the Achyla steroid receptor, cross-reacted with Achlya hsp85 in cytosols from heat-shocked cells. This monoclonal antibody also recognized both the hormone-induced and heat shock-induced 85-kDa in vitro translation products. Taken together, these data suggest that similar or identical 85-kDa proteins are independently regulated by the steroid hormone antheridiol and by heat shock and that this protein is part of the Achyla steroid receptor complex. Our results demonstrate that the association of hsp90 family proteins with steroid receptors observed in mammals and birds extends also to the eucaryotic microbes and suggest that this association may have evolved early in steroid-responsive systems.     

The 5' untranslated region of Rhopalosiphum padi virus contains an internal ribosome entry site which functions efficiently in mammalian, plant, and insect translation systems

Rhopalosiphum padi virus (RhPV) is one of several picorna-like viruses that infect insects; sequence analysis has revealed distinct differences between these agents and mammalian picornaviruses. RhPV has a single-stranded positive-sense RNA genome of about 10 kb; unlike the genomes of Picornaviridae, however, this genome contains two long open reading frames (ORFs). ORF1 encodes the virus nonstructural proteins, while the downstream ORF, ORF2, specifies the structural proteins. Both ORFs are preceded by long untranslated regions (UTRs). The intergenic UTR is known to contain an internal ribosome entry site (IRES) which directs non-AUG-initiated translation of ORF2. We have examined the 5' UTR of RhPV for IRES activity by translating synthetic dicistronic mRNAs containing this sequence in a variety of systems. We now report that the 5' UTR contains an element which directs internal initiation of protein synthesis from an AUG codon in mammalian, plant, and Drosophila in vitro translation systems. In contrast, the encephalomyocarditis virus IRES functions only in the mammalian system. The RhPV 5' IRES element has features in common with picornavirus IRES elements, in that no coding sequence is required for IRES function, but also with cellular IRES elements, as deletion analysis indicates that this IRES element does not have sharply defined boundaries.     

An adenovirus mRNA which encodes a 14,700-Mr protein that maps to the last open reading frame of region E3 is expressed during infection.

The E3 regions of adenovirus types 2 and 5, respectively, are known to synthesize proteins of 19,000 Mr (19K) and 11.6K, but information regarding the identity and characterization of other potential E3 proteins encoded by the six remaining open reading frames (ORFs) is lacking. In this study, we show that the last ORF of region E3, which encodes a 14.7K protein, is expressed in adenovirus-infected cells. This information was largely derived from analysis of an E3 deletion mutant (H2dl801) in which an extensive deletion (1,939 base pairs) was found to eliminate all ORFs except for two proteins of 12.5K and 14.7K. The 14.7K protein was translated from RNA isolated from H2dl801-infected cells that had been hybridization selected to E3 DNA; hybridization-selected RNA from wild-type adenovirus type 5-infected cells translated both the 19K and the 14.7K proteins. Moreover, an antiserum directed against a bacterial 14.7K fusion protein (A. E. Tollefson and W. S. M. Wold, J. Virol. 62:33-39, 1988) immunoprecipitated the 14.7K translation product synthesized by wild-type and mutant H2dl801 adenovirus mRNAs.     

A heat shock-resistant mutant of Saccharomyces cerevisiae shows constitutive synthesis of two heat shock proteins and altered growth

A heat shock-resistant mutant of the budding yeast Saccharomyces cerevisiae was isolated at the mutation frequency of 10(-7) from a culture treated with ethyl methane sulfonate. Cells of the mutant are approximately 1,000-fold more resistant to lethal heat shock than those of the parental strain. Tetrad analysis indicates that phenotypes revealed by this mutant segregated together in the ratio 2+:2- from heterozygotes constructed with the wild-type strain of the opposite mating type, and are, therefore, attributed to a single nuclear mutation. The mutated gene in the mutant was herein designated hsr1 (heat shock response). The hsr1 allele is recessive to the HSR1+ allele of the wild-type strain. Exponentially growing cells of hsr1 mutant were found to constitutively synthesize six proteins that are not synthesized or are synthesized at reduced rates in HSR1+ cells unless appropriately induced. These proteins include one hsp/G0-protein (hsp48A), one hsp (hsp48B), and two G0-proteins (p73, p56). Heterozygous diploid (hsr1/HSR1+) cells do not synthesize the proteins constitutively induced in hsr1 cells, which suggests that the product of the HSR1 gene might negatively regulate the synthesis of these proteins. The hsr1 mutation also led to altered growth of the mutant cells. The mutation elongated the duration of G1 period in the cell cycle and affected both growth arrest by sulfur starvation and growth recovery from it. We discuss the problem of which protein(s) among those constitutively expressed in growing cells of the hsr1 mutant is responsible for heat shock resistance and alterations in the growth control.     

Selective translation of heat shock mRNA in Drosophila melanogaster depends on sequence information in the leader.

One of the effects of a temperature increase above 35 degrees C on Drosophila melanogaster is a rapid switch in selectivity of the translational apparatus. Protein synthesis from normal, but not from heat shock, mRNA is much reduced. Efficient translation at high temperature might be a result of the primary sequence of heat shock genes. Alternatively a mRNA modification mechanism, altered as a consequence of heat shock, might allow for efficient high temperature translation of any mRNA synthesized during a heat shock. The gene for alcohol dehydrogenase (Adh) was fused to the controlling elements of a heat shock protein 70 (hsp70) gene. Authentic Adh mRNA, synthesized from this fusion gene at elevated temperatures was not translated during heat shock. A second Adh fusion gene in which the mRNA synthesized contained the first 95 nucleotides of the Hsp70 non-translated leader sequence gave rise, at high temperature, to mRNA which was translated during the heat shock. Thus, the signal(s) in the mRNAs controlling translation efficiency at heat shock temperatures is encoded within the heat shock genes.