Efficient intracellular delivery of an exogenous protein GFP with genetically fused basic oligopeptides.

Several oligopeptides, derived from certain proteins, translocate as a form fused to small molecules or exogenous proteins across the plasma membrane into cells. Some of these oligopeptides, the so-called protein-transduction domains (PTDs), contain a high proportion of basic residues. The translocation of some of these basic PTDs, such as oligoarginines, has been studied as chemically fused forms to other organic compounds. In this study, we also tested to determine whether or not oligoarginines, when fused genetically to an exogenous protein such as GFP, are also able to translocate efficiently across the plasma membrane. The oligoarginine Rn (n = 5,6,7,8,9)-GFP fusion proteins were translocated quite efficiently, and the transduction efficiency increased in proportion to the number of arginine residues. However, the cellular uptake of the oligolysine-GFP fusion proteins was less efficient than that of the corresponding oligoarginine-GFP fusion proteins. When fused to GFP, the translocation efficiency of R5 was similar to that of Tat(49-57)(RKKRRQRRR). This finding suggests that the arginine homo-oligopeptide is more efficient than other PTDs which contain a mixture of basic residues. On the other hand, both the K9- and Tat(49-57)-GFP fusion proteins were transduced with similar efficiencies. It appears that basic oligopeptides may be useful for the efficient translocation of diverse exogenous proteins as genetically fused forms.     

Baboon endogenous virus genome. I. Restriction enzyme map of the unintegrated DNA genome of a primate retrovirus

A detailed restriction map was deduced for the genome of an endogenous retrovirus of a higher primate, that of baboon. The cleavage sites for 12 restriction enzymes were mapped. The unintegrated linear viral DNA intermediate that is produced by infection of permissive cells with baboon endogenous virus was isolated. Hybridization with a strong-stop complementary DNA probe demonstrated presence of a terminal repetition in the linear viral DNA. The positions of restriction sites for two particular enzymes, SmaI and XhoI, near each end were consistent with this result and indicated that the length of the repetition is 0.55 +/- 0.01 kilobase. The linear viral DNA had a unique restriction map indicating that it is not a set of random circular permutations of the RNA genome. From hybridization with a 3'-specific probe, the DNA restriction map was aligned relative to the 5'-to-3' orientation of the viral RNA. We observed a minor heterogeneity in a BamHI recognition site 1.95 kilobases from the right end of the linear map.     

LINE drive. retrotransposition and genome instability

The LINE-1 (L1) retrotransposon, the most important human mobile element, shapes the genome in many ways. Now two groups provide evidence that L1 retrotransposition is associated with large genomic deletions and inversions in transformed cells. If these events occur at a similar frequency in vivo, they have had a substantial effect on human genome evolution.     

Fluidity of a retrovirus genome.

Comparison of the genomic sequences of the Friend spleen focus-forming virus with other murine retroviral sequences indicated that the spleen focus-forming virus was derived from at least three retroviruses. The 5' end of the virus, from the primer binding site through most of gag, was derived from AKV. The rest of gag and all of pol were of uncertain origin, but were probably derived from the same xenotropic virus that gave rise to the 5' half of env. The remainder was derived from Friend murine leukemia virus. The positions of a 585-base deletion, a 6-base duplication, and a point insertion that leads to a frame shift and premature protein termination in the ecotropic 3' end of env were invariant between three spleen focus-forming virus strains, indicating that they had a single common ancestor. However, the point of crossover between xenotropic viral sequences and Friend murine leukemia virus was different in each strain, and two strains were much more closely related to each other than to the third in the xenotropic region, indicating that these strains had diverged by multiple recombinations. Furthermore, a different nucleotide comprised the single point insertion near the 3' end of env, suggesting that these viruses have an extremely high transition and transversion rate.     

Squirrel monkey retrovirus: an endogenous virus of a new world primate.

Squirrel monkey retrovirus (SMRV) was isolated by cocultivation of squirrel monkey lung cells with canine cells. 3H-labeled 60-70S SMRV RNA, isolated from virus grown in canine cells, hybridized to the same extent and to the same Cot1/2 value to the DNA of all tissues of all squirrel monkeys tested; Cot1/2 values show that SMRV proviral sequences are present in the low repetitive range. No SMRV proviral sequences were detected in tissues from a variety of other New World monkeys, Old World monkeys, or apes. Murine, feline, bovine, and canine cells also contain no detectable SMRV proviral sequences. Competitive molecular hybridization studies revealed no detectable sequence homology between the 60-70S RNAs of SMRV and Mason-Pfizer virus (MPV). The virion-associated DNA polymerase of SMRV is similar to that of MPV in that it has a molecular weight of approximately 80,000 and prefers magnesium as a divalent cation using oligo(dG)-poly(rC) as primer-template. The virion-associated DNA polymerase of SMRV can be clearly distinguished from those of MPV and the mouse mammary tumor viruses, however, by its preference for manganese as a divalent cation in the presence of high salt.     

Molecular cloning of the unintegrated squirrel monkey retrovirus genome: organization and distribution of related sequences in primate DNAs

The closed circular form of the endogenous squirrel monkey type D retrovirus (SMRV) was molecularly cloned in a bacteriophage vector. The restriction map of the biologically active clone was determined and found to be identical to that of the parental SMRV linear DNA except for the deletion of one long terminal repeat. Restriction enzyme analysis and Southern blotting indicated that the SMRV long terminal repeat was approximately 300 base pairs long. The SMRV restriction map was oriented to the viral RNA by using a gene-specific probe from baboon endogenous virus. Restriction enzyme digests of a variety of vertebrate DNAs were analyzed for DNA sequence homology with SMRV by using the cloned SMRV genome as a probe. Consistent with earlier studies, multiple copies of SMRV were detected in squirrel monkey DNA. Related fragments were also detected in the DNAs from other primate species, including humans.     

Infection of nonhuman primate cells by pig endogenous retrovirus

The ongoing shortage of human donor organs for transplantation has catalyzed new interest in the application of pig organs (xenotransplantation). One of the biggest concerns about the transplantation of porcine grafts into humans is the transmission of pig endogenous retroviruses (PERV) to the recipients or even to other members of the community. Although nonhuman primate models are excellently suited to mimic clinical xenotransplantation settings, their value for risk assessment of PERV transmission at xenotransplantation is questionable since all of the primate cell lines tested so far have been found to be nonpermissive for PERV infection. Here we demonstrate that human, gorilla, and Papio hamadryas primary skin fibroblasts and also baboon B-cell lines are permissive for PERV infection. This suggests that a reevaluation of the suitability of the baboon model for risk assessment in xenotransplantation is critical at this point.     

Nucleotide sequence of the jaagsiekte retrovirus, an exogenous and endogenous type D and B retrovirus of sheep and goats.

The complete genome of the jaagsiekte sheep retrovirus (JSRV), the suspected etiological agent of ovine pulmonary carcinoma, has been cloned from viral particles secreted in lung exudates of affected animals and sequenced. The genome is 7,462 nucleotides long and exhibits a genetic organization characteristic of the type B and D oncoviruses. Comparison of the amino acid sequences of JSRV proteins with those of other retrovirus proteins and phylogenetic studies suggest that JSRV diverged from its type B and D lineage after the type B mouse mammary tumor virus but before the type D oncoviruses captured the env gene of a reticuloendotheliosislike virus. Southern blot studies show that closely related sequences are present in sheep and goat normal genomic DNA, indicating that JSRV could be endogenous in ovine and caprine species.     

Transfection by exogenous and endogenous murine retrovirus DNAs.

A low molecular weight (LMW) protein phosphokinase enzyme that binds to actin has been isolated from murine sarcoma virions; this kinase activity is not present in nontransforming murine leukemia viruses. Sephadex G-75 gel filtration and affinity chromatography on actin-Sepharose conjugates allow a significant level of purification of this enzyme. The enzyme associates with microtubular proteins and inhibits the in vitro polymerization of microtubules. This study represents the first isolation of a sarcoma virus-associated protein that possesses the ability to interact directly with two major components of the cytoskeletal system.     

Characterization of the bovine endogenous retrovirus beta3 genome

We recently used degenerate PCR and locus-specific PCR methods to identify the endogenous retroviruses (ERV) in the bovine genome. Using the ovine ERV classification system, the bovine ERVs (BERVs) could be classified into four families. Here, we searched the most recently released bovine genome database with the partial nucleotide sequence of the pro/pol region of the BERV beta3 family. This allowed us to obtain and analyze the complete genome of BERV beta3. The BERV beta3 genome is 7666 nucleotides long and has the typical retroviral organization, namely, 5'-long terminal repeat (LTR)-gag-pro-pol-env-LTR-3'. The deduced open reading frames for gag, pro, pol and env of BERV Beta en- code 507, 271, 879 and 603 amino acids, respectively. BERV beta3 showed little amino acid similarity to other betaretroviruses. Phylogenetic analysis showed that it clusters with HERV-K. This is the first report describing the genetic structure and sequence of an entire BERV.     

Moving primate genomics beyond the chimpanzee genome

The comparative DNA sequence data that already exist on individual genomic loci depict the phylogenetic relationships of nearly all extant primate genera. Such a phylogenetic representation of the primates, validated by many sequenced primate genomes, and encompassing the full adaptive diversity of the order, is a prerequisite for identifying the genetic basis of humankind, and for testing the proposed human uniqueness of these traits. Some of these traits have been discovered recently, particularly in genes encoding proteins that are important for brain function.     

Efficient multiple genome alignment

MOTIVATION: To allow a direct comparison of the genomic DNA sequences of sufficiently similar organisms, there is an urgent need for software tools that can align more than two genomic sequences. RESULTS: We developed new algorithms and a software tool 'Multiple Genome Aligner' (MGA for short) that efficiently computes multiple genome alignments of large, closely related DNA sequences. For example, it can align 85% percent of the complete genomes of six human adenoviruses (average length 35305 bp.) in 159 seconds. An alignment of 74% of the complete genomes of three of strains of E. coli (lengths: 5528445; 5498450; 4639221 approximately bp.) is produced in 30 minutes.     

A substrate ambiguous enzyme facilitates genome reduction in an intracellular symbiont

Background

Genome evolution in intracellular microbial symbionts is characterized by gene loss, generating some of the smallest and most gene-poor genomes known. As a result of gene loss these genomes commonly contain metabolic pathways that are fragmented relative to their free-living relatives. The evolutionary retention of fragmented metabolic pathways in the gene-poor genomes of endosymbionts suggests that they are functional. However, it is not always clear how they maintain functionality. To date, the fragmented metabolic pathways of endosymbionts have been shown to maintain functionality through complementation by host genes, complementation by genes of another endosymbiont and complementation by genes in host genomes that have been horizontally acquired from a microbial source that is not the endosymbiont. Here, we demonstrate a fourth mechanism.

Results

We investigate the evolutionary retention of a fragmented pathway for the essential nutrient pantothenate (vitamin B5) in the pea aphid, Acyrthosiphon pisum endosymbiosis with Buchnera aphidicola. Using quantitative analysis of gene expression we present evidence for complementation of the Buchnera pantothenate biosynthesis pathway by host genes. Further, using complementation assays in an Escherichia coli mutant we demonstrate functional replacement of a pantothenate biosynthesis enzyme, 2-dehydropantoate 2-reductase (E.C. 1.1.1.169), by an endosymbiont gene, ilvC, encoding a substrate ambiguous enzyme.

Conclusions

Earlier studies have speculated that missing enzyme steps in fragmented endosymbiont metabolic pathways are completed by adaptable endosymbiont enzymes from other pathways. Here, we experimentally demonstrate completion of a fragmented endosymbiont vitamin biosynthesis pathway by recruitment of a substrate ambiguous enzyme from another pathway. In addition, this work extends host/symbiont metabolic collaboration in the aphid/Buchnera symbiosis from amino acid metabolism to include vitamin biosynthesis.