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Mitochondrial heat shock protein 70 (mtHsp70) functions in unfolding, translocation, and folding of imported proteins. Controversial models of mtHsp70 action have been discussed: (1) physical trapping of preproteins is sufficient to explain the various mtHsp70 functions, and (2) unfolding of preproteins requires an active motor function of mtHsp70 ("pulling"). Intragenic suppressors of a mutant mtHsp70 separate two functions: a nonlethal folding defect caused by enhanced trapping of preproteins, and a conditionally lethal unfolding defect caused by an impaired interaction of mtHsp70 with the membrane anchor Tim44. Even enhanced trapping in wild-type mitochondria does not generate a pulling force. The motor function of mtHsp70 cannot be explained by passive trapping alone but includes an essential ATP-dependent interaction with Tim44 to generate a pulling force and unfold preproteins.

The function of the yeast SSB 70 kd heatshock proteins (hsp70s) was investigated by a variety of approaches. The SSB hsp70s (Ssb1/2p) are associated with translating ribosomes. This association is disrupted by puromycin, suggesting that Ssb1/2p may bind directly to the nascent polypeptide. Mutant ssb1 ssb2 strains grow slowly, contain a low number of translating ribosomes, and are hypersensitive to several inhibitors of protein synthesis. The slow growth phenotype of ssb1 ssb2 mutants is suppressed by increased copy number of a gene encoding a novel translation elongation factor 1 alpha (EF-1 alpha)-like protein. We suggest that cytosolic hsp70 aids in the passage of the nascent polypeptide chain through the ribosome in a manner analogous to the role played by organelle-localized hsp70 in the transport of proteins across membranes.

In this paper we describe the isolation and characterization of a 7.2 kb D. melanogaster chromosomal DNA fragment (K1) which contains nucleotide sequences complementary to D. melanogaster actin mRNA. Plasmid K1 was identified using a Dictyostelium actin cDNA plasmid, B1, as a probe. D. melanogaster mRNA selected by hybridization with immobilized K1 DNA was translated in vitro to yield products which co-migrate with the D. melanogaster actins I, II and III in two-dimensional gel electrophoresis and bind to DNAase I agarose. A physical map localizing restriction endonuclease cleavage sites in the K1 DNA fragment and the direction of transcription is presented. The position of the coding region has been localized by hybridization with labeled B1 DNA and with labeled poly(A)-containing D. melanogaster RNA. On the basis of hybridization of labeled subfragments of plasmid K1 to restriction endonuclease-cleaved D. melanogaster embryo DNA, we conclude that the nucleotide sequence of the presumptive coding region is responsible for labeling of a pattern of multiple restriction fragments from embryo DNA. The chromosomal locus from which DNA fragment K1 is derived has been localized by in situ hybridization to two closely linked bands in the region 88F. Related DNA sequences corresponding to putative actin genes have also been mapped cytologically. These results support the hypothesis that the genes for actin in D. melanogaster are members of a closely related family of coding sequences.

S. cerevisiae contains a family of genes related to the major heat shock induced gene of Drosophila (Hsp70). Two members of this family, YG100 and YG102, are 97% identical to each other in their protein coding regions. RNA transcribed from YG100 increases markedly after a heat shock, while transcripts of YG102 increase minimally. Mutants of the two genes were constructed in vitro and substituted in the yeast genome in place of the wild-type alleles. No phenotypic effect of single mutations of either gene was detected. However, cells containing both the YG100 and YG102 mutations grew slowly at 30 degrees C and could not form colonies at 37 degrees C. The temperature sensitive phenotype can be overcome by inserting either an intact YG100 or YG102 gene into the genome. Pretreatment at 37 degrees C before shift to a normally lethal temperature of 51 degrees C protected the double mutant as well as the wild type, indicating that YG100 and YG102 gene products are not needed for resistance to high temperatures for short periods.

We have isolated recombinant DNA clones which include cDNA and chromosomal DNA sequences of the major heat shock-inducible gene of Drosophila. With the cDNA fragments used as specific hybridization probes, DNA:DNA reassociation and in situ hybridization analysis demonstrated that the DNA sequences are repeated approximately 7 times in the haploid Drosophila genome, and that gene sequences are present at both the 87A and 87C loci on the cytological map. The cloned cDNA and homologous cloned chromosomal DNA hybridized to mRNA which translated in vitro into the major 70K heat shock-specific protein. Here we summarize a study of the organization of genes coding for the 70K heat shock-specific protein contained in the two recombinant chromosomal DNA plasmids pG3 and pG5. On the basis of R loop hybridization experiments and restriction enzyme analysis, we conclude that a 14 kb fragment, G3, contains three copies of the gene coding for the 70K protein. A second 9.2 kb fragment, G5, contains one copy of the gene coding for the 70K protein. Hybridization of labeled poly(A)-containing RNA to restriction endonuclease-cleaved DNA indicates that the mRNA coding regions in G3 and G5 are each approximately 2100 bp long. The three tandemly repeated genes of G3 are separated by approximately 1400 bp of spacer DNA. The two internal spacer regions in G3 appear to be identical, whereas differences in restriction enzyme sites indicate that the sequences adjacent to the cluster differ from the internal spacer and from each other.

The primary sequence of the major heat shock gene of D. melanogaster, that for the 70,000 protein, has been determined. One of the reading frames is devoid of stop codons for over 2000 bp. The region between the first ATG and the first stop codon encodes a protein of molecular weight 70,270. The 5' end of the messenger RNA was localized in the DNA sequence by two independent methods. The 5' flanking sequences of three distinct 70K genes were also determined. Extensive homology in the primary sequences extends about 500 bp upstream from the ATG, which is the presumptive initiation of protein synthesis. Each 70K gene has the putative promoter sequence TATAAATA about 325 bp upstream from this ATG. A heptanucleotide sequence identified as the capping site for other messengers is found 24-30 bp downstream from the ends of the A-T-rich sequence. A 12 bp sequence with dyad symmetry begins 23 bp upstream from the beginning of the above A-T-rich sequence.

Nuclear viral RNAs synthesized early in productive infection with adenovirus 2 were analyzed by hybridization to specific viral DNA fragments. Radioactive RNAs were subjected to electrophoresis in polyacrylamide gels containing 98% formamide, and the fractionated RNA was hybridized to specific DNA fragments generated by cleavage with endo R-Eco Ri or endo R-Sma l. The viral genes expressed early in infection are located in four different segments of the genome. When nuclear RNA was hybridized to DNA fragments representing each of these segments, discrete RNA size classes were detected. For each of these four regions of the genome, some of the discrete nuclear RNAs were larger than the cytoplasmic mRNAs. As calculated from electrophoretic mobility, the molecular weight of these nuclear RNA size classes ranged from 15% to several fold greater than that of the corresponding cytoplasmic mRNAs. Hybridization-inhibition experiments were performed to analyze the sequences present in one of these larger nuclear transcripts, a 22S RNA transcribed from the left-hand end of the genome. This 22S nuclear RNA was transcribed from the same strand as the viral mRNA, for it contained cytoplasmic sequences; it also contained sequences restricted to the nucleus. The larger nuclear RNAs may serve as precursors to the cytoplasmic viral mRNAs synthesized early in infection.