Amino-terminal amino acid sequence of the major structural polypeptides of avian retroviruses: sequence homology between reticuloendotheliosis virus p30 and p30s of mammalian retroviruses.

The major structural polypeptides, p30 of reticuloendotheliosis virus (REV) (strain T) and p27 of avian sarcoma virus B77, have been compared with regard to amino acid composition. NH2-terminal amino acid sequence, and immunological crossreactions. The amino acid composition of the two polypeptides is distinct, and a comparison of the first 30 NH2-terminal amino acids of REV p30 with that for the first 25 of B77 p27 yields only three homologous residues. In competition radioimmunoassays the polypeptides show no crossreactivity. A comparison of the amino acid composition and NH2-terminal amino acid sequence of REV p30 with those reported for several mammalian retrovirus p30s shows remarkable similarities. Both REV and mammalian p30s contain a large number of polar residues in their amino acid composition and show approximately 40% homology in the first 30 NH2-terminal amino acids. No crossreactivity could be observed, however, in competition radioimmunoassays between Rauscher murine leukemia virus p30 and that of REV. The observations reported here suggest a close evolutionary relationship between REV and the mammalian retroviruses.     

Phorbol ester-induced expression of primate retroviruses (author's transl)

Addition of the tumor promoter 12-0-Tetradecanoyl-phorbol-13-acetate (TPA) to the growth medium of certain human cell cultures persistently infected with simian retroviruses of type C (BaEV, SSV) or type D (MPMV) or a human cell line-derived type D isolate (PMFV), respectively, resulted in a considerable but transient stimulation of virus production. Enhanced virus expression was paralleled by striking morphological alterations of the cells. Among four infected cell types tested so far, ony one (A 204) failed to respond to TPA with significant virus stimulation or altered morphology.     

Interspecies antigenic determinants of structural proteins of mammalian type-C viruses as detected by a competitive sepharose bead immunofluorescence assay.

The present paper describes a competitive immunoassay using antiserum to FeLV p 27 and SSV-conjugated Sepharose beads. The assay is applied to compare the interspecies-specific antigenic determinants of the major structural proteins of type-C viruses of different mammalian species. The test proves to be highly sensitive and specific and may be used for the demonstration of viral proteins in crude cellular extracts.     

A general model for the transmembrane proteins of HIV and other retroviruses

A hypothetical model of the transmembrane (TM) protein of human immunodeficiency virus (HIV) is proposed that is derived from the known structure of the influenza TM protein HA2. This model is consistent with computer algorithms of predicted protein structure and with known properties of TM proteins determined by sequence homology, site-directed mutations, peptide analogs, immunochemistry, or other biologic means. It is applicable to a wide variety of retroviral TM proteins differing widely in overall molecular weight.     

Common antigenic determinants on structural proteins p10--12 of PMFV and MPMV type D retroviruses

PMFV, a type D retrovirus isolated from a malignant human embryo cell line, was compared with Mason-Pfizer monkey virus (MPMV) in a sensitive tannic acid enhanced indirect immunodiffusion test. In addition to the previously shown common antigens, both viruses contain identical group-specific antigenic determinants on their p 10--12 as demonstrated with a specific p 10--12 MPMV test system. Interspecies mammalian type C virus antigens were not detected in highly concentrated PMFV preparations.     

New chromosomal location for structural genes of ribosomal proteins.

An Escherichia coli mutant, ts9, previously reported by Flaks et al. (Cold Spring Harbor Symp. Quant. Biol. 31, 623-631, 1966) to have an electrophoretically altered ribosomal protein, has been further characterized and the altered component has been identified as L7/L12. Although mutant ts9 is temperature sensitive for growth (rts-), the rts and L7/L12 mutations are genetically separable and are both located between argH and rif. The L7/L12 mutation (rpyL) maps very close to relC, mutants of which have a defect in the 50S ribosomal subunit. The gene order is argH-rts-(rpyL,relC)-rif. Protein synthesis directed by bacteriophage lambdacI857S7drifd18 in ultraviolet-irradiated cells indicates that L7/L12, As well as L1, L10, L11, and possibly L8 or L9 are coded by the phage DNA. Our results indicate that rpyL is the structural gene for L7/L12 and that this region of the E. coli chromosome contains a cluster of structural genes for ribosomal proteins.     

Mapping the structural genes coding for the major urinary proteins in the mouse: combined use of recombinant inbred strains and somatic cell hybrids.

We have mapped the multiple (15-25) genes coding for the hormonally regulated major urinary proteins (MUPs) of the mouse by using a cloned cDNA probe. By Southern blot analysis of DNA from Chinese hamster-mouse somatic cell hybrids, all of the MUP genes were found to be on chromosome 4. Different inbred mouse strains showed DNA polymorphism in their MUP Southern hybridization pattern. Analysis of recombinant inbred strains derived from these parent strains has shown that all the polymorphisms are linked to the MUP-a locus on chromosome 4. The combination of these mapping techniques should be applicable to many cloned DNA sequences.     

Invariance and heterogeneity in the major structural and regulatory proteins of chick muscle cells revealed by two-dimensional gel electrophoresis.

A two-dimensional gel electrophoresis system is used to investigate some of the properties of desmin, the major subunit of the 100-A filaments from chick muscle cells, and to compare these properties to those of the other major contractile and regulatory proteins of muscle. Desmin from embryonic and adult smooth, skeletal, and cardiac muscle cells is resolved into two isoelectric variants, alpha and beta, which possess slightly different electrophoretic mobilities in sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Both the alpha and the beta variants from all six preparations appear to be identical in isoelectric point and apparent molecular weight. The alpha and beta desmin are present in approximately equal amounts in all three types of muscle, suggesting that both isoelectric variants of desmin serve as the structural subunits of the 100-A filaments in chick muscle cells. Tropomyosin also can be resolved into two subunits, alpha and beta, in all three types of muscle. However, in each type of muscle both subunits differ from their counterparts in the other types of muscle, either by molecular weight or by isoelectric point. These results indicate that, with regard to apparent isoelectric point and molecular weight, desmin, a major muscle structural protein, is invariant, while tropomyosin, a major muscle regulatory protein, exhibits heterogeneity in the three types of muscle.     

A radioimmunoassay for erythropoietin.

We describe the development of a radioimmunoassay (RIA) for erythropoietin. Antisera were raised in rabbits with an impure human urinary erythropoietin preparation used as immunogen, but with pure human erytropoietin serving as the labeled antigen in the RIA and as a primary standard. The immunoreactivity of erythropoietin is not altered significantly by the mode of labeling with radioiodine, even though the biologic activity is lost. With this method, it is possible to detect 2--3 mU of erythropoietin in a volume of 0.1--0.3 ml. Therefore, the method can be used for detection of normal and subnormal serum titers as well as elevated titers. RIA for erythropoietin does not distinguish between the native (active in vivo) and the asialo form (inactive in vivo) and cannot yet be used for routine monitoring of crude fractions during purification.     

Interspecies homology of nitrogenase genes.

Cloned nitrogen fixation (nif) genes from Klebsiella pneumoniae hybridize to DNA from 19 out of 19 widely divergent nitrogen-fixing bacterial strains but do not hybridize to DNA from 10 different non-nitrogen-fixing species. K. pneumoniae nif DNA fragments that hybridize to DNA from other species contain part of the three structural genes that code for nitrogenase polypeptides. We have utilized this homology to clone an EcoRI restriction endonuclease fragment from Rhizobium meliloti that hybridizes to the K. pneumoniae nif structural genes. Some of the species whose DNA hybridizes with K. pneumoniae nif DNA have been postulated to have diverged from K. pneumoniae 3 x 10(9) years ago. Nitrogenase genes are the only known example of such highly conserved prokaryotic translated genes. Nitrogenase genes are either extraordinarily conserved in evolution or have been exchanged between different nitrogen-fixing species relatively recently in evolutionary time.     

Teleocidin, a tumour promoter, stimulates synthesis of primate retroviruses in persistently infected human cells

The naturally occurring tumour promoter teleocidin produces a pronounced but transient enhancement of the synthesis of extracellular viral particles in human cells chronically infected with simian retroviruses of type C (baboon endogenous virus, simian sarcoma virus) or type D (Mason-Pfizer monkey virus) or a human cell line-derived type D isolate (PMF virus), respectively. The retrovirus-stimulating activity of teleocidin is very similar to that previously described for the tumour-promoting phorbol ester TPA in the same cell systems.     

Nuclear matrins: identification of the major nuclear matrix proteins.

A preparative two-dimensional polyacrylamide gel system was used to separate and purify the major Coomassie blue-stained proteins from the isolated rat liver nuclear matrix. Approximately 12 major proteins were consistently found. Of these, 5 proteins represented identified proteins, including nuclear lamins A, B, and C, the nucleolar protein B-23, and residual components of core heterogeneous nuclear ribonucleoproteins. The remaining eight major proteins termed the nuclear matrins consisted of matrin 3 (125 kDa, slightly acidic), matrin 4 (105 kDa, basic), matrins D-G (60-75 kDa, basic), and matrins 12 and 13 (42-48 kDa, acidic). Peptide mapping and two-dimensional immunoblot studies indicate that matrins D-G compose two pairs of related proteins (matrins D/E and F/G) and that none of the matrins resemble the nuclear lamins or any of the other major proteins detected on our two-dimensional gels. Subfractionation immunoblot experiments demonstrated the nearly exclusive localization of matrins F/G and other matrins to the nuclear matrix fraction of the cell. These results were further supported by indirect immunofluorescence microscopy that showed a strictly interior nuclear localization of the matrins in intact cells in contrast to the peripherally located nuclear lamins. We conclude that the nuclear matrins are a major class of proteins of the nuclear matrix interior and are distinct from the nuclear lamins.     

Amino-terminal sequence analysis of the structural proteins of Sindbis virus.

The structural proteins of Sindbis virus, an enveloped virus which belongs to the Togavirus family, have been subjected to automated Edman degradation using improved techniques. Extensive NH2-terminal sequences of about 50 residues were determined for each of the two membrane glycoproteins. In both cases the NH2 terminus of the molecule was found to be similar in composition to typical water-soluble proteins. The viral capsid protein was found to have a blocked alpha-amino group. This is consistent with other observations that viral proteins derived from the NH2 terminus of precursor molecules are often blocked.     

Binding of buried structural water increases the flexibility of proteins.

Water deeply buried in proteins is considered to be an integral part of the folded structure. Such structural water molecules make strong H bonds with polar groups of the surrounding protein and therefore are believed to tighten the protein matrix. Surprisingly, our computational analysis of the binding of a buried water molecule to bovine pancreatic trypsin inhibitor shows that the protein actually becomes more flexible, as revealed by an increase in the vibrational entropy. We find that this effect must be common in proteins, because the large entropic cost of immobilizing a single water molecule [-TDeltaS = 20.6 kcal/mol (1 kcal = 4.18 kJ) for the lost translational and rotational degrees of freedom] can only be partly compensated by water-protein interactions, even when they are nearly perfect, as in the case of bovine pancreatic trypsin inhibitor (DeltaE = -19.8 kcal/mol), leaving no room for a further decrease in entropy from protein tightening. This study illustrates the importance of considering changes in protein flexibility (which in this case favor binding by 3.5 kcal/mol) for the prediction of ligand binding affinities.     

Sperm-associated retroviruses in the mouse epididymis.

Sperm adsorbed with retrovirus particles were recovered from the epididymis of apparently normal male mice. Epididymal semen from all four mouse strains examined was positive for retrovirus (10(5) to 10(8) particles per microgram of protein) indicating that epididymal fluids and sperm may be important vehicles for murine retrovirus spread. Immunoblot analyses revealed that the banding patterns of electrophoretically separated epididymal viral proteins from the four strains of males were more similar to each other than to either xenotropic New Zealand Black virus or ecotropic Rauscher leukemia virus proteins. The results indicate that retrovirus particles, possibly a unique strain, are commonly expressed at relatively high titers in the reproductive tract of male mice and are sperm-associated.     

Homologous radioimmunoassay for canine prolactin.

A homologous double antibody radioimmunoassay has been developed for canine prolactin. Purified canine prolactin was iodinated by lactoperoxidase/H2O2 to an average of 101 "+/- 10.1 muCi/microgram. Antiserum was used at a final dilution of 1:80 000 and at this concentration bound approximately 20% of the added tracer in the absence of competing unlabelled prolactin. Partial cross-reaction was observed with ovine and bovine prolactin but there was no cross-reaction with a highly purified canine growth hormone preparation. Dilutions of pregnant and lactating bitch sera were parallel to the purified canine prolactin standard curve. Mean prolactin levels in normal male and anoestrous females were 8.8 +/- 0.8 and 12.6 +/- 2.6 ng/ml, respectively. Thyrotrophin-releasing hormone (TRH) induced a consistent elevation in prolactin levels 15--30 min after intravenous injection.