Complete nucleotide sequence, genome organization, and biological properties of human immunodeficiency virus type 1 in vivo: evidence for limited defectiveness and complementation.

Previous studies of the genetic and biologic characteristics of human immunodeficiency virus type 1 (HIV-1) have by necessity used tissue culture-derived virus. We recently reported the molecular cloning of four full-length HIV-1 genomes directly from uncultured human brain tissue (Y. Li, J. C. Kappes, J. A. Conway, R. W. Price, G. M. Shaw, and B. H. Hahn, J. Virol. 65:3973-3985, 1991). In this report, we describe the biologic properties of these four clones and the complete nucleotide sequences and genome organization of two of them. Clones HIV-1YU-2 and HIV-1YU-10 were 9,174 and 9,176 nucleotides in length, differed by 0.26% in nucleotide sequence, and except for a frameshift mutation in the pol gene in HIV-1YU-10, contained open reading frames corresponding to 5'-gag-pol-vif-vpr-tat-rev-vpu-env-nef-3' flanked by long terminal repeats. HIV-1YU-2 was fully replication competent, while HIV-1YU-10 and two other clones, HIV-1YU-21 and HIV-1YU-32, were defective. All three defective clones, however, when transfected into Cos-1 cells in any pairwise combination, yielded virions that were replication competent and transmissible by cell-free passage. The cellular host range of HIV-1YU-2 was strictly limited to primary T lymphocytes and monocyte-macrophages, a property conferred by its external envelope glycoprotein. Phylogenetic analyses of HIV-1YU-2 gene sequences revealed this virus to be a member of the North American/European HIV-1 subgroup, with specific similarity to other monocyte-tropic viruses in its V3 envelope amino acid sequence. These results indicate that HIV-1 infection of brain is characterized by the persistence of mixtures of fully competent, minimally defective, and more substantially altered viral forms and that complementation among them is readily attainable. In addition, the limited degree of genotypic heterogeneity observed among HIV-1YU and other brain-derived viruses and their preferential tropism for monocyte-macrophages suggest that viral replication within the central nervous system may differ from that within the peripheral lymphoid compartment in significant and clinically important ways. The availability of genetically and biologically well characterized HIV-1 clones from uncultured human tissue should facilitate future studies of virus-cell interactions relevant to viral pathogenesis and drug and vaccine development.     

Conjugative gene transfer in marine cyanobacteria: Synechococcus sp., Synechocystis sp. and Pseudanabaena sp.

Summary Versatility of gene transfer by transconjugation in marine cyanobacteria was demonstrated. In this study, seven different marine cyanobacteria were used as recipient cells. First, transconjugation was carried out using the mobilizable transposon (Tn5) carrying plasmid pSUP1021. Transconjugants were observed in all marine cyanobacteria tested. Second, the broad-host-range vector pKT230 (IncQ) was tested for transconjugation. pKT230 has been successfully transferred in a marine cyanobacterium Synechococcus sp. NKBG15041C, and replicated as an autonomous replicon without alteration in the restriction enzyme pattern. A maximum transfer efficiency of 5.2 × 10^–4 transconjugants/recipient cell was observed, when mating was performed on agar plates containing low salinity (0.015 m NaCl) medium. This is the first study to demonstrate gene transfer in marine cyanobacteria via transconjugation. Correspondence to: K. Sode     

Effect of sterol side-chain structure on the feed-back control of sterol biosynthesis in yeast

We measured the incorporation of radiolabeled methionine and acetate into the sterol component of G204, a Saccharomyces cerevisiae mutant strain which is partially heme competent. By comparing the amount of label incorporated into the sterol pool of a control culture, to which no exogenous sterol was added, with a culture which had various sterols added to the growth medium, we were able to determine the specific structural features of ergosterol which facilitate its ability to restrict the sterol biosynthetic pathway. These experiments demonstrate that sterols which contain both a C22 unsaturation and a C24 methyl group are capable of reducing sterol biosynthesis by approx. 50%, regardless of B-ring structure. We examined the regulatory properties of various oxysterols; 24,25-epoxylanosterol reduced endogenous biosynthesis by 49%, whereas all cholesterol derivatives tested, including 25-hydroxycholesterol, had little effect. A new procedure for the synthesis of ergosterol peroxides is also described.     

Resistance to receptor-mediated degradation of a murine epidermal growth factor analogue (EGF-Val-47) potentiates its mitogenic activity

In most cell types two classes of epidermal growth factor (EGF) receptors can be found: a major class that binds EGF with relatively low affinity and a minor class that binds with very high affinity. Structure-function studies have shown that mutations at amino acid 47 in the EGF molecule severely reduce its affinity for the EGF receptor but do not cause preferential binding to one or the other subclass of receptors. Using three EGF derivatives with a mutation at amino acid 47 (Ser-47, Leu-37-Tyr-47, and Val-47), we have investigated the relative contribution of the two receptor subclasses to the EGF-dependent mitogenic response. We show that mitogenicity correlates exclusively with occupancy of the high-affinity receptor and that full occupancy of this subclass is required for maximal stimulation. In addition we demonstrate that for the EGF-Val-47 analogue this requirement can be abrogated and half-maximal biological activity reached with a high-affinity receptor occupancy of only 8%. While the rate of internalization did not significantly differ between EGF-Val-47 and native mEGF, the analogue was much more resistant to degradation by cellular proteases and, after binding and receptor-mediated internalization, was released into the medium predominantly in an intact form. We propose that the increased mitogenicity of EGF-Val-47 is due to its prolonged half-life, resulting in continued occupancy of the high-affinity EGF receptor.     

Sequence identity between the alpha 2-macroglobulin receptor and low density lipoprotein receptor-related protein suggests that this molecule is a multifunctional receptor.

Ten peptides, derived from human alpha 2-macroglobulin (alpha 2M) receptor by chemical or proteolytic digestion, were sequenced. Comparative analysis revealed that all of the resulting sequences were present within the cDNA-deduced structure of low density lipoprotein receptor-related protein (LRP) (Herz, J., Hamann, U., Rogne, S., Myklebost, O., Gausepohl, H., and Stanley, K. K. (1988) EMBO J. 7, 4119-4127). The findings provide evidence that the alpha 2M receptor and LRP are the same molecule. Further evidence comes from immunoprecipitation experiments using a monoclonal antibody specific for the alpha 2M receptor that show this molecule, like LRP, to contain two polypeptides of approximately 420 and 85 kDa that are noncovalently associated. An additional component of this receptor system is a 39-kDa polypeptide that co-purifies with the alpha 2M receptor during affinity chromatography. Solid phase binding studies reveal that the 39-kDa polypeptide binds with high affinity (Kd = 18 nM) to the 420-kDa component of the alpha 2M receptor. The apparent identity of LRP and the alpha 2M receptor suggests that this molecule is a multifunctional receptor with the capacity to bind diverse biological ligands and highlights a possible relationship between two previously unrelated biological processes, lipid metabolism and proteinase regulation.     

Purification of eukaryotic RNA polymerase II by immunoaffinity chromatography. Elution of active enzyme with protein stabilizing agents from a polyol-responsive monoclonal antibody

Active eukaryotic RNA polymerase II (RNAP II) was purified by immunoaffinity chromatography, using a monoclonal antibody (mAb) that reacts with the highly conserved heptapeptide repeat of the largest subunit. This mAb (designated SWG16) was conjugated to CNBr-activated Sepharose and used to purify RNAP II from wheat germ and calf thymus. The subunit composition of the immunoaffinity-purified enzyme was essentially the same as RNAP II purified by conventional chromatography except that it contained only the form with the unproteolyzed largest subunit. Active enzyme could be eluted from the SWG16-Sepharose, at pH 7.9, with combinations of low molecular weight polyols and nonchaotropic salts. The superior eluting procedure used combinations of ethylene glycol (30-40%) and ammonium sulfate (0.5-0.75 M). Active enzyme also could be eluted with a synthetic peptide containing four repeats of the heptapeptide; however, the peptide was not as effective as the polyol and salt combinations for eluting the enzyme. This mAb should be useful for purifying RNAP II from many eukaryotic species. Because the elution of enzyme from the immunoadsorbent seems to be dependent upon the presence of a polyol, this antibody is referred to as a "polyol-responsive mAb." A procedure that helps to identify a polyol-responsive mAb and to optimize the eluting conditions is described. Polyol-responsive mAbs might have broad applicability to the purification of many labile enzymes by immunoaffinity chromatography.     

Concepts of occlusion: Australian evidence

Longitudinal studies of aboriginal children over a 20-year period have drawn attention to the wide variation in morphological features of the dentition and the way in which occlusal relationships develop. This paper summarizes some important determinants of optimal occlusal development, namely, tooth size relationships within and between dentitions, the patterns of alveolar growth, and tooth migrations during the transition from primary to permanent teeth and the nature of growth changes in the dental arches. Dental occlusion constantly changes throughout life in response to changing functional requirements. Observations limited to cross-sectional material provide an incomplete, and sometimes misleading, concept of dental occlusion and masticatory function.