Roy J. Britten, 1919–2012: Our early years at Caltech

Roy Britten died in Costa Mesa, California on January 21, 2012, of pancreatic cancer at age 92. His work in the 1960s, in which he used renaturation kinetics to provide a quantitative image of the single-copy and repetitive sequence content of animal genomes, was of gigantic intellectual import, and it essentially built the ground floor of the edifice that we call genomics today. He was elected a member of the National Academy of Sciences in 1972. At the beginning of the 1970s, Roy and I teamed up as scientific partners, and we relocated to Caltech. At Caltech, we worked together for over one-quarter of a century, and most of the following work consists of a very brief retrospective on the eventful first decade of our Caltech partnership. Later, in the 1990s, Roy returned to focus on his old interests in evolutionary processes that affect genomic sequence content. He continued to carry out computational analyses on the roles of mobile elements and other processes that ceaselessly remodel genomes, particularly primate genomes, almost until his death; his last paper, “Transposable element insertions have strongly affected human evolution,” was published in PNAS in November of 2010 when he was 91 years old

The refolding activity of the yeast heat shock proteins Ssa1 and Ssa2 defines their role in protein translocation.

Ssa1/2p, members of one of the yeast cytosolic hsp70 subfamilies, have been implicated in the translocation of secretory proteins into the lumen of the ER. The involvement of these hsp70s in translocation was tested directly by examining the effect of immunodepleting Ssa1/2p from yeast cytosol and subsequently testing the cytosol for its ability to support co- and post-translational translocation of prepro-alpha-factor. Depletion of Ssa1/2p had no effect on the efficiency of translocation in this in vitro assay. The system was used to examine the effect of the absence of Ssa1/2p on two other putative hsp70 functions: cotranslational folding of nascent luciferase and refolding of denatured luciferase. Depletion of Ssa1/2p had no effect on the ability of the yeast lysate to synthesize enzymatically active luciferase, but had a dramatic effect on the ability of the lysate to refold chemically denatured luciferase. These results demonstrate, for the first time, the refolding activity of Ssa1/2p in the context of the yeast cytosol, and define refolding activity as a chaperone function specific to Ssa1/2p, aprt from other cytosolic hsp70s. They also suggest that Ssa1/2p do not play a significant role in chaperoning the folding of nascent polypeptides. The implications of these findings for Ssa1/2p activity on their proposed role in the process of translocation are discussed

An acidic region of the 89K murine cytomegalovirus immediate early protein interacts with DNA

The product of the ie 1 gene, the regulatory immediate early protein pp89 of murine cytomegalovirus (MCMV), interacts with core histones, which can mediate the association of pp89 with DNA. We report the capacity of pp89 to interact directly with DNA in the absence of cellular proteins. After separation of proteins by SDS–PAGe, pp89 bound ds- and ssDNA, with a preference for ssDNA. Binding to specific DNA sequences in the MCMV genome was not detected. The DNA-binding region of pp89 was located to amino acids 438 to 534 by analysis of deletion mutants expressed as -galactosidase or TrpE fusion proteins. This region is identical to the highly acidic C-terminal region spanning amino acids 424 to 532. The human cytomegalovirus IE1 protein, which contains a similar extended C-terminal acidic region, does not react with DNA under the same experimental conditions

Pseudomonas chloritidismutans sp. nov., a non-denitrifying chlorate-reducing bacterium

A Gram-negative, facultatively anaerobic, rod-shaped, dissimilatory chlorate-reducing bacterium, strain AW-1(T), was isolated from biomass of an anaerobic chlorate-reducing bioreactor. Phylogenetic analysis of the 16S rDNA sequence showed 100␜equence similarity to Pseudomonas stutzeri DSM 50227 and 98.6␜equence similarity to the type strain of P. stutzeri (DSM 5190(T)). The species P. stutzeri possesses a high degree of genotypic and phenotypic heterogeneity. Therefore, eight genomic groups, termed genomovars, have been proposed based upon DeltaT(m) values, which were used to evaluate the quality of the pairing within heteroduplexes formed by DNA--DNA hybridization. In this study, DNA--DNA hybridization between strain AW-1(T) and P. stutzeri strains DSM 50227 and DSM 5190(T) revealed respectively 80.5 and 56.5␜imilarity. DNA--DNA hybridization between P. stutzeri strains DSM 50227 and DSM 5190(T) revealed 48.4␜imilarity. DNA--DNA hybridization indicated that strain AW-1(T) is not related at the species level to the type strain of P. stutzeri. However, strain AW-1(T) and P. stutzeri DSM 50227 are related at the species level. The physiological and biochemical properties of strain AW-1(T) and the two P. stutzeri strains were compared. A common characteristic of P. stutzeri strains is the ability to denitrify. However, in growth experiments, strain AW-1(T) could use only chlorate or oxygen as an electron acceptor and not nitrate, perchlorate or bromate. Strain AW-1(T) is the first chlorate-reducing bacterium described that does not possess another oxyanion-reduction pathway. Cell extracts of strain AW-1(T) showed chlorate and bromate reductase activities but not nitrate reductase activity. P. stutzeri strains DSM 50227 and DSM 5190(T) could use nitrate or oxygen as an electron acceptor, but not chlorate. Chlorate reductase activity, in addition to nitrate reductase activity, was detected in cell extracts of both P. stutzeri strains. Chlorite dismutase activity was absent in extracts of both P. stutzeri strains but was present in extracts of strain AW-1(T). Based on the hybridization experiments and the physiological and biochemical data, it is proposed that strain AW-1(T) be classified as a novel species of Pseudomonas, Pseudomonas chloritidismutans sp. nov. The type strain is strain AW-1(T) (=DSM 13592(T)=ATCC BAA-443(T))

DNA unwinding component of the nonhistone chromatin proteins

A subclass of nonhistone chromatin proteins from rat liver, previously shown to exhibit high affinity for DNA, has been fractionated by single-stranded DNA-agarose affinity chromatography. The protein fraction that bound to DNA-agarose in 0.19 M NaCl-buffer and was eluted with 2 M NaCl-buffer is enriched for a protein component of approximately 20,000 daltons and exhibits preferential binding to denatured DNA. This nonhistone protein fraction specific for single strands binds to DNA in a non-species-specific manner, and causes helix-coil transition of synthetic poly[d(A-T)· d(A-T)] at 25 degrees, as indicated by the increase in absorbance of ultraviolet light at 260 nm. The observed hyperchromicity does not result from any nuclease activity in the protein fraction, because addition of Mg+2 results in partial hypochromic shift, and the protein/DNA complex is retained by nitrocellulose filters

Interactions of Cisplatin and Daunorubicin at the chromatin level

Unexpectedly, the widely used anticancer agents Cisplatin (Cis-Pt) and Daunorubicin (Dauno) exhibited cell type- and concentration-dependent synergy or antagonism in vitro. We attempted to interpret these effects in terms of the changes elicited by the drugs in the chromatin, the target held primarily responsible for the cytotoxicity of both agents. We measured the effect of Cis-Pt on the levels of Dauno in different cell compartments, the effect of Cis-Pt on Dauno-induced nucleosome eviction, and assessed the influence of Dauno on DNA platination in flow- and laser scanning cytometry as well as in laser ablation-inductively coupled plasma-mass spectrometry assays. We show that the two drugs antagonize each other through a decrease of interstrand crosslinks upon co-treatment with Dauno, and also via the diminished Dauno uptake in the presence of Cis-Pt, and both effects are observed already at low Dauno concentrations. At high Dauno concentrations synergy becomes dominant because histone eviction by Dauno intercalation into the DNA is enhanced in the presence of co-treatment with Cis-Pt. These interactions may have an impact on the efficacy of combination treatment protocols, considering the long retention time of DNA adducts formed by both agents

Encoding folding paths of RNA switches

RNA co-transcriptional folding has long been suspected to play an active role in helping proper native folding of ribozymes and structured regulatory motifs in mRNA untranslated regions. Yet, the underlying mechanisms and coding requirements for efficient co-transcriptional folding remain unclear. Traditional approaches have intrinsic limitations to dissect RNA folding paths, as they rely on sequence mutations or circular permutations that typically perturb both RNA folding paths and equilibrium structures. Here, we show that exploiting sequence symmetries instead of mutations can circumvent this problem by essentially decoupling folding paths from equilibrium structures of designed RNA sequences. Using bistable RNA switches with symmetrical helices conserved under sequence reversal, we demonstrate experimentally that native and transiently formed helices can guide efficient co-transcriptional folding into either long-lived structure of these RNA switches. Their folding path is controlled by the order of helix nucleations and subsequent exchanges during transcription, and may also be redirected by transient antisense interactions. Hence, transient intra- and intermolecular base pair interactions can effectively regulate the folding of nascent RNA molecules into different native structures, provided limited coding requirements, as discussed from an information theory perspective. This constitutive coupling between RNA synthesis and RNA folding regulation may have enabled the early emergence of autonomous RNA-based regulation networks.Comment: 9 pages, 6 figure