A heterologous, high-affinity RNA ligand for human immunodeficiency virus Gag protein has RNA packaging activity.

Retroviral RNA encapsidation depends on the specific binding of Gag proteins to packaging (psi) signals in genomic RNA. We investigated whether an in vitro-selected, high-affinity RNA ligand for the nucleocapsid (NC) portion of the Gag protein from human immunodeficiency virus type 1 (HIV-1) could mediate packaging into HIV-1 virions. We find that this ligand can functionally substitute for one of the Gag-binding elements (termed SL3) in the HIV-1 psi locus to support packaging and viral infectivity in cis. By contrast, this ligand, which fails to dimerize spontaneously in vitro, is unable to replace a different psi element (termed SL1) which is required for both Gag binding and dimerization of the HIV-1 genome. A single point mutation within the ligand that eliminates high-affinity in vitro Gag binding also abolishes its packaging activity at the SL3 position. These results demonstrate that specific binding of Gag or NC protein is a critical determinant of genomic RNA packaging.

In vitro selection of RNAs that bind to the human immunodeficiency virus type-1 gag polyprotein.

RNA ligands that bind to the human immunodeficiency virus type-1 (HIV-1) gag polyprotein with 10(-9) M affinity were isolated from a complex pool of RNAs using an in vitro selection method. The ligands bind to two different regions within gag, either to the matrix protein or to the nucleocapsid protein. Binding of a matrix ligand to gag did not interfere with the binding of a nucleocapsid ligand, and binding of a nucleocapsid ligand to gag did not interfere with the binding of a matrix ligand. However, binding of a nucleocapsid ligand to gag did interfere with binding of an RNA containing the HIV-1 RNA packaging element (psi), even though the sequence of the nucleocapsid ligand is not similar topsi. The minimal sequences required for the ligands to bind to matrix or nucleocapsid were determined. Minimal nucleocapsid ligands are predicted to form a stem-loop structure that has a self-complementary sequence at one end. Minimal matrix ligands are predicted to form a different stem-loop structure that has a CAARU loop sequence. The properties of these RNA ligands may provide tools for studying RNA interactions with matrix and nucleocapsid, and a novel method for inhibiting HIV replication.

Homologous and unique G protein alpha subunits in the nematode Caenorhabditis elegans.

A cDNA corresponding to a known G protein alpha subunit, the alpha subunit of Go (Go alpha), was isolated and sequenced. The predicted amino acid sequence of C. elegans Go alpha is 80-87% identical to other Go alpha sequences. An mRNA that hybridizes to the C. elegans Go alpha cDNA can be detected on Northern blots. A C. elegans protein that crossreacts with antibovine Go alpha antibody can be detected on immunoblots. A cosmid clone containing the C. elegans Go alpha gene (goa-1) was isolated and mapped to chromosome I. The genomic fragments of three other C. elegans G protein alpha subunit genes (gpa-1, gpa-2, and gpa-3) have been isolated using the polymerase chain reaction. The corresponding cosmid clones were isolated and mapped to disperse locations on chromosome V. The sequences of two of the genes, gpa-1 and gpa-3, were determined. The predicted amino acid sequences of gpa-1 and gpa-3 are only 48% identical to each other. Therefore, they are likely to have distinct functions. In addition they are not homologous enough to G protein alpha subunits in other organisms to be classified. Thus C. elegans has G proteins that are identifiable homologues of mammalian G proteins as well as G proteins that appear to be unique to C. elegans. Study of identifiable G proteins in C. elegans may result in a further understanding of their function in other organisms, whereas study of the novel G proteins may provide an understanding of unique aspects of nematode physiology.

Cone cell-specific genes expressed in retinoblastoma.

Retinoblastoma, an intraocular tumor that occurs in children, has long been regarded, on the basis of morphological criteria, as a malignancy of the photoreceptor cell lineage. Here it is shown that when this tumor is grown in vitro, the cells express highly specialized photoreceptor cell genes. Transcripts for the transducin alpha subunit, TC alpha, which is specific to the cone cell, as well as transcripts for the red or green cone cell photopigment, were found in seven out of seven low-passage retinoblastoma cell lines. No marker genes specific to rod cell were expressed, suggesting that retinoblastoma has a cone cell lineage.

The nuclear-coded subunits of yeast cytochrome c oxidase. The amino acid sequences of subunits VII and VIIa, structural similarities between the three smallest polypeptides of the holoenzyme, and implications for biogenesis.

The complete amino acid sequences of subunits VII and VIIa from yeast cytochrome c oxidase are reported. Subunits VII and VIIa are 57 residues (Mr = 6603) and 54 residues (Mr = 6303) in length, respectively. Both polypeptides are amphiphilic, have an internal hydrophobic section and hydrophilic NH2 and COOH termini, and terminate at their COOH termini with a basic amino acid. This structural motif is similar to that possessed by subunit VIII of yeast cytochrome c oxidase. All three polypeptides have hydrophobic sections which are long enough to span the inner membrane; all three polypeptides lack methionine at their NH2 termini; and all three polypeptides have COOH termini which could result from proteolysis by a protease with trypsin or cathepsin B-like activity. These observations raise the interesting possibility that subunits VII, VIIa, and VIII are transmembranous polypeptides which are processed at both their NH2 and COOH termini during their biogenesis.

Sequence of the alpha subunit of photoreceptor G protein: homologies between transducin, ras, and elongation factors.

A bovine retinal complementary DNA clone encoding the alpha subunit of transducin (T alpha) was isolated with the use of synthetic oligodeoxynucleotides as probes, and the complete nucleotide sequence of the insert was determined. THe predicted protein sequence of 354 amino acids includes the known sequences of four tryptic peptides and sequences adjacent to the residues that undergo adenosine diphosphate ribosylation by cholera toxin and pertussis toxin. On the basis of homologies to other proteins, such as the elongation factors of protein synthesis and the ras oncogene proteins, regions are identified that are predicted to be acylated and involved in guanine nucleotide binding and hydrolysis. Amino acid sequence similarity between T alpha and ras is confined to these regions of the molecules.

The nuclear-coded subunits of yeast cytochrome c oxidase. I. Fractionation of the holoenzyme into chemically pure polypeptides and the identification of two new subunits using solvent extraction and reversed phase high performance liquid chromatography.

Previously, cytochrome c oxidase from the yeast Saccharomyces cerevisiae has been thought to be composed of seven different polypeptide subunits. Four of these are small polypeptides (4,000-15,000 daltons), subunits IV-VII, which are encoded by nuclear DNA. Studies described here reveal the presence of two new polypeptides in this size range. These polypeptides, designated as subunits VIIa and VIII, co-migrate with subunit VII (R.O. Poyton and G. Schatz (1975) J. Biol. Chem. 250, 752-761) on low resolution sodium dodecyl sulfate (SDS) polyacrylamide gels, can be partially resolved on high resolution SDS polyacrylamide gels, and can be completely separated from one another by reversed phase high performance liquid chromatography. In order to determine the sequences of each of these six nuclear-coded polypeptides (subunits IV, V, VI, VII, VIIa, and VIII), we have developed new methods for the large scale purification of the holoenzyme and have employed a new strategy for the isolation of each polypeptide. By using octyl-Sepharose chromatography to isolate holocytochrome c oxidase and by extracting the holoenzyme with aprotic organic solvents and fractionating these extracts by reversed phase high performance liquid chromatography, it is possible to isolate several milligrams of each of these subunits. Each subunit preparation gives a single peak during reversed phase high performance liquid chromatography, a single band during SDS-polyacrylamide gel electrophoresis, a single NH2-terminal sequence, and a unique amino acid composition and tryptic peptide map. Since each purified subunit preparation gives close to a 100% yield of its NH2-terminal amino acid during quantitative Edman degradation, we conclude that no subunit has a blocked NH2 terminus and that no subunit preparation contains either blocked or unblocked contaminating polypeptides. Thus, each consists of a single unique polypeptide species. Together, these results demonstrate that yeast cytochrome c oxidase contains six, rather than four, small subunit polypeptides. Thus, it appears that these polypeptides, in combination with the three polypeptides encoded by mitochondrial DNA, constitute a holoenzyme which contains nine subunits, instead of seven as proposed earlier.

The nuclear-coded subunits of yeast cytochrome c oxidase. II. The amino acid sequence of subunit VIII and a model for its disposition in the inner mitochondrial membrane.

The amino acid sequence of subunit VIII from yeast cytochrome c oxidase is reported. This 47-residue (Mr = 5364) amphiphilic polypeptide has a polar NH2 terminus, a hydrophobic central section, and a dilysine COOH terminus. An analysis of local hydrophobicity and predicted secondary structure along the peptide chain predicts that the hydrophobic central region is likely to be transmembranous. Subunit VIII from yeast cytochrome c oxidase exhibits 40.4% homology to bovine heart cytochrome c oxidase subunit VIIc , at the level of primary structure. Secondary structures and hydrophobic domains predicted from the sequences of both polypeptides are also highly conserved. From the location of hydrophobic domains and the positions of charged amino acid residues we have formulated a topological model for subunit VIII in the inner mitochondrial membrane.

The nuclear-coded subunits of yeast cytochrome c oxidase. III. Identification of homologous subunits in yeast, bovine heart, and Neurospora crassa cytochrome c oxidases.

Sequences for the NH2-terminal halves of subunits IV, V, VI, VII, and VIIa from yeast cytochrome c oxidase have been determined and used to identify homologous subunits in bovine heart and Neurospora crassa cytochrome c oxidases. In conjunction with the complete sequence of subunit VIII (S. D. Power, M. A. Lochrie , T. E. Patterson, and R. O. Poyton (1984) J. Biol. Chem. 259, 6571-6574), we have been able to identify counterparts to yeast subunits IV, V, VI, and VIII in bovine heart cytochrome c oxidase and counterparts to yeast subunits IV and V in Neurospora crassa cytochrome c oxidase. The sequences of these nuclear-coded subunits are conserved between species at a level of 30-50%. Thus, they are conserved to the same extent as the three mitochondrially coded subunits (I, II, and III). The similar degree of homology between species for both the nuclear and mitochondrially coded subunits of cytochrome c oxidase suggests that both sets of polypeptides are conserved coordinately and are, therefore, important components of the functional holoenzyme.

Reversed-phase high-performance liquid chromatographic purification of subunits of oligomeric membrane proteins. The nuclear coded subunits of yeast cytochrome c oxidase.

Reversed-phase chromatography of the subunits of an oligomeric membrane protein such as yeast cytochrome c oxidase requires additional sample handling techniques which are not necessary for soluble proteins. This paper considers these and discusses (1) methods for the removal of ballast material by preliminary batchwise extraction with solvent mixtures similar to those used for reversed-phase elution; (2) the chromatographic heterogeneity induced by partial cysteine oxidation; (3) the removal of tightly bound proteins from the stationary phase; and (4) the generation of an elution system with continuously variable selectivity based on acetonitrile-1-propanol ratios (0.05% triethylamine, 0.05% trifluoroacetic acid). These methods are designed to simplify complex mixtures of hydrophobic proteins prior to chromatography and to purify them chromatographically in high yield.