Mark G. Rush

Characterization of four novel ras-like genes expressed in a human teratocarcinoma cell line.

A mixed-oligonucleotide probe was used to identify four ras-like coding sequences in a human teratocarcinoma cDNA library. Two of these sequences resembled the rho genes, one was closely related to H-, K-, and N-ras, and one shared only the four sequence domains that define the ras gene superfamily. Homologs of the four genes were found in genomic DNA from a variety of mammals and from chicken. The genes were transcriptionally active in a range of human cell types.

Cellular functions of TC10, a Rho family GTPase: regulation of morphology, signal transduction and cell growth.

The small Ras-related GTPase, TC10, has been classified on the basis of sequence homology to be a member of the Rho family. This family, which includes the Rho, Rac and CDC42 subfamilies, has been shown to regulate a variety of apparently diverse cellular processes such as actin cytoskeletal organization, mitogen-activated protein kinase (MAPK) cascades, cell cycle progression and transformation. In order to begin a study of TC10 biological function, we expressed wild type and various mutant forms of this protein in mammalian cells and investigated both the intracellular localization of the expressed proteins and their abilities to stimulate known Rho family-associated processes. Wild type TC10 was located predominantly in the cell membrane (apparently in the same regions as actin filaments), GTPase defective (75L) and GTP-binding defective (31N) mutants were located predominantly in cytoplasmic perinuclear regions, and a deletion mutant lacking the carboxyl terminal residues required for post-translational prenylation was located predominantly in the nucleus. The GTPase defective (constitutively active) TC10 mutant: (1) stimulated the formation of long filopodia; (2) activated c-Jun amino terminal kinase (JNK); (3) activated serum response factor (SRF)-dependent transcription; (4) activated NF-κB-dependent transcription; and (5) synergized with an activated Raf-kinase (Raf-CAAX) to transform NIH3T3 cells. In addition, wild type TC10 function is required for full H-Ras transforming potential. We demonstrate that an intact effector domain and carboxyl terminal prenylation signal are required for proper TC10 function and that TC10 signals to at least two separable downstream target pathways. In addition, TC10 interacted with the actin-binding and filament-forming protein, profilin, in both a two-hybrid cDNA library screen, and an in vitro binding assay. Taken together, these data support a classification of TC10 as a member of the Rho family, and in particular, suggest that TC10 functions to regulate cellular signaling to the actin cytoskeleton and processes associated with cell growth.

Evolutionary implications of primate endogenous retroviruses

Endogenous DNA sequences related to retroviruses are probably present in all primates. By using approaches based on the polymerase chain reaction, two separate studies have revealed the evolutionary history of some of these sequences. In the first study, a retrovirus-like reverse transcriptase (RT) sequence homologous to that of Baboon endogenous virus (BaEV) has been identified in both Old World monkeys and African apes, but not in humans or Asian apes. This RT sequence is highly conserved at the amino acid level, but not the nucleotide level, in the baboon, African green monkey, Java macaque, chimpanzee, and gorilla. The patterns of nucleotide substitution indicate functional conservation and suggest that this RT sequence was present in the primate germline before apes and Old World monkeys diverged about 30 million years ago. In the second study, a comparison of endogenous proviral DNAs and their adjacent sequences has been used to analyze the evolutionary history of three previously reported human endogenous retroviruses, HERV-E(4.14), HERV-R(3), and HERV-Ia. It is shown that these retroviruses have also been resident in the primate line since before the ape-Old World monkey divergence. The implications of the presence of functionally conserved RT genes in the germlines of primates, and the potential for using integration sites as tools for analyzing phylogenetic relationships among primates and their retroviruses, are discussed.

Effects of mutant Ran/TC4 proteins on cell cycle progression.

Ran/TC4, a member of the RAS gene superfamily, encodes an abundant nuclear protein that binds and hydrolyzes GTP. Transient expression of a Ran/TC4 mutant protein deficient in GTP hydrolysis blocked DNA replication, suggesting a role for Ran/TC4 in the regulation of cell cycle progression. To test this possibility, we exploited an efficient transfection system, involving the introduction of cDNAs in the pMT2 vector into 293/Tag cells, to analyze phenotypes associated with mutant and wild-type Ran/TC4 expression. Expression of a Ran/TC4 mutant protein deficient in GTP hydrolysis inhibited proliferation of transfected cells by arresting them predominantly in the G2, but also in the G1, phase of the cell cycle. Deletion of an acidic carboxy-terminal hexapeptide from the Ran/TC4 mutant did not alter its nuclear localization but did block its inhibitory effect on cell cycle progression. These data suggest that normal progression of the cell cycle is coupled to the operation of a Ran/TC4 GTPase cycle. Mediators of this coupling are likely to include the nuclear regulator of chromosome condensation 1 protein and the mitosis-promoting factor complex.

Separate domains of the Ran GTPase interact with different factors to regulate nuclear protein import and RNA processing.

The small Ras-related GTP binding and hydrolyzing protein Ran has been implicated in a variety of processes, including cell cycle progression, DNA synthesis, RNA processing, and nuclear-cytosolic trafficking of both RNA and proteins. Like other small GTPases, Ran appears to function as a switch: Ran-GTP and Ran-GDP levels are regulated both by guanine nucleotide exchange factors and GTPase activating proteins, and Ran-GTP and Ran-GDP interact differentially with one or more effectors. One such putative effector, Ran-binding protein 1 (RanBP1), interacts selectively with Ran-GTP. Ran proteins contain a diagnostic short, acidic, carboxyl-terminal domain, DEDDDL, which, at least in the case of human Ran, is required for its role in cell cycle regulation. We show here that this domain is required for the interaction between Ran and RanBP1 but not for the interaction between Ran and a Ran guanine nucleotide exchange factor or between Ran and a Ran GTPase activating protein. In addition, Ran lacking this carboxyl-terminal domain functions normally in an in vitro nuclear protein import assay. We also show that RanBP1 interacts with the mammalian homolog of yeast protein RNA1, a protein involved in RNA transport and processing. These results are consistent with the hypothesis that Ran functions directly in at least two pathways, one, dependent on RanBP1, that affects cell cycle progression and RNA export, and another, independent of RanBP1, that affects nuclear protein import.

Extrachromosomal DNA in eucaryotes

Eucaryotic extrachromosomal DNAs have been organized into four major classes: (1) Organelle DNAs, (2) plasmid DNAs, (3) amplified genes, and (4) intermediates and/or by-products of DNA transpositions and rearrangements. In this review some of the relatively well-characterized members of each class are described; it is suggested that many of them reflect the complexity and plasticity of eucaryotic genomes.

Members of the Alu family of interspersed, repetitive DNA sequences are in the small circular DNA population of monkey cells grown in culture.

Small, polydisperse circular DNA isolated from the BSC-1 line of African Green Monkey kidney cells was shown, by cross hybridization with an Alu repetitive DNA probe, to contain sequences homologous to the Alu family of mobile, dispersed repetitive DNA sequences. The circular nature of these molecules was demonstrated by two independent techniques. In addition Alu sequences were detected in a bank of cloned spc-DNA. The nucleotide sequence for one of these clones was determined and found to be homologous to 83% of a human Alu consensus sequence. The absence of short direct repeats, which usually flank Alu repetitive DNA sequence elements, is consistent with a variety of models in which Alu -containing spc-DNAs represent intermediates in the movement of Alu -dispersed genetic elements between chromosomal sites.

Tissue-specific expression of Ran isoforms in the mouse

Ran genes encode a family of well-conserved small nuclear GTPases (Ras-related nuclear proteins), whose function is implicated in both normal cell cycle progression and the transport of RNA and proteins between the nucleus and the cytoplasm. Previous studies of Ran proteins have utilized cell-free systems, yeasts, and cultured mammalian cells. We have now characterized patterns of Ran gene expression in the mouse. Serum starvation suppressed Ran gene transcription in mouse 3T3 cells. Ran mRNA reappeared in cells within 3 h after refeeding. A single Ran mRNA species was detected at low levels in most somatic tissues of the adult mouse. In testis, this Ran mRNA was abundant, as were other larger transcripts. Analysis of testis-derived Ran cDNA clones revealed the presence of two transcripts, one specifying an amino acid sequence identical to that of human Ran/TC4 and one specifying an amino acid sequence 94% identical. Northern blotting and reverse transcriptase-PCR assays with oligonucleotide probes and primers specific for each transcript demonstrated that the isoform identical to Ran/TC4 was expressed in both somatic tissues and testis, while the variant form was transcribed only in testis. The existence of tissue-specific Ran isoforms may help to rationalize the diverse roles suggested for Ran by previous biochemical studies.

Structure of extrachromosomal circular DNAs containing both the Alu family of dispersed repetitive sequences and other regions of chromosomal DNA

Small polydisperse circular DNA (spcDNA) isolated from the BSC-1 line of African Green monkey kidney cells was digested with the restriction endonuclease BamHI and cloned in bacteriophage lambda. The resulting library of 25,000 phage was then screened for the presence of the Alu family of short interspersed nucleotide sequences, and four of the 100 Alu-positive clones were characterized. In summary: (1) all four clones contained regions other than Alu that were homologous to the BSC-1 chromosome. Two contained Alu plus unique chromosomal DNA, one contained Alu plus an uncharacterized repetitive chromosomal DNA, and one contained Alu plus both unique and a specific tandemly repeated chromosomal DNA (alpha-satellite); (2) all four clones were derived from extrachromosomal circular DNAs and not from the accidental cloning of a very small amount of contaminating chromosomal material assumed to be present in spcDNA preparations; and (3) one clone represented an intact circular DNA with a restriction endonuclease cleavage map that was a circularly permuted version of its chromosomal homologue.

Isolation and preliminary characterization of the small circular DNA present in African green monkey kidney (BSC-1) cells.

Abstract The small polydisperse circular DNA (spc-DNA) previously identified in SV40-infected African green monkey kidney (BSC-1) cells ( M. G. Rush, R. Eason, and J. Vinograd, 1971 , Biochim. Biophys. Acta 228, 585–594.) has been isolated in pure form from uninfected cells. This double-stranded, covalently closed circular DNA contains species ranging in molecular weight from about 0.1 to 4 × 106, although most of the molecules are distributed in an apparently polydisperse population with molecular weights of less than 1 × 106. There are approximately 1000 to 2000 covalently closed small DNA molecules per cell, and their average buoyant density does not appear to differ significantly from that of chromosomal and mitochondrial DNAs. This spc-DNA was resolved by polyacrylamide gel electrophoresis into three distinct bands containing comparatively homogeneous circular DNAs with molecular weights of 200,000, 520,000, and 780,000. However, the reassociation rate of in vitro labeled, denatured spc-DNA suggested a molecular complexity in the range of 1 × 108, and the ability of BSC-1 chromosomal DNA to accelerate greatly the reassociation of about one third of this material indicated the presence of some repetitive chromosomal DNA sequences in spc-DNA.