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Biomedical subjects

F Gao

Publications and source records attributed to F Gao.

At least 19 recordsLinked to original sources
PubMed1999

The 20 C-terminal amino acid residues of the chloroplast ATP synthase gamma subunit are not essential for activity.

It has been suggested that the last seven to nine amino acid residues at the C terminus of the gamma subunit of the ATP synthase act as a spindle for rotation of the gamma subunit with respect to the alpha beta subunits during catalysis (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628). To test this hypothesis we selectively deleted C-terminal residues from the chloroplast gamma subunit, two at a time starting at the sixth residue from the end and finishing at the 20th residue from the end. The mutant gamma genes were overexpressed in Escherichia coli and assembled with a native alpha3beta3 complex. All the mutant forms of gamma assembled as effectively as the wild-type gamma. Deletion of the terminal 6 residues of gamma resulted in a significant increase (>50%) in the Ca-dependent ATPase activity when compared with the wild-type assembly. The increased activity persisted even after deletion of the C-terminal 14 residues, well beyond the seven residues proposed to form the spindle. Further deletions resulted in a decreased activity to approximately 19% of that of the wild-type enzyme after deleting all 20 C-terminal residues. The results indicate that the tip of the gammaC terminus is not essential for catalysis and raise questions about the role of the C terminus as a spindle for rotation.

Amino Acid Sequence↗
PubMed1999

Inhibition of p38 mitogen-activated protein kinase decreases cardiomyocyte apoptosis and improves cardiac function after myocardial ischemia and reperfusion.

BACKGROUND: Activation of p38 mitogen-activated protein kinase (MAPK) plays an important role in apoptotic cell death. The role of p38 MAPK in myocardial injury caused by ischemia/reperfusion, an extreme stress to the heart, is unknown. METHODS AND RESULTS: Studies were performed with isolated, Langendorff-perfused rabbit hearts. Ischemia alone caused a moderate but transient increase in p38 MAPK activity (3.5-fold increase, P<0.05 versus basal). Ischemia followed by reperfusion further activated p38 MAPK, and the maximal level of activation (6.3-fold, P<0.01) was reached 10 minutes after reperfusion. Administration of SB 203580, a p38 MAPK inhibitor, decreased myocardial apoptosis (14.7+/-3.2% versus 30.6+/-3.5% in vehicle, P<0.01) and improved postischemic cardiac function. The cardioprotective effects of SB 203580 were closely related to its inhibition of p38 MAPK. Administering SB 203580 before ischemia and during reperfusion completely inhibited p38 MAPK activation and exerted the most cardioprotective effects. In contrast, administering SB 203580 10 minutes after reperfusion (a time point when maximal MAPK activation had already been achieved) failed to convey significant cardioprotection. Moreover, inhibition of p38 MAPK attenuated myocardial necrosis after a prolonged reperfusion. CONCLUSIONS: These results demonstrate that p38 MAPK plays a pivotal role in the signal transduction pathway mediating postischemic myocardial apoptosis and that inhibiting p38 MAPK may attenuate reperfusion injury.

Animals↗
PubMed1999

Q-Band resonance Raman investigation of turnip cytochrome f and Rhodobacter capsulatus cytochrome c1.

The results of a comprehensive Q-band resonance Raman investigation of cytochrome c1 and cytochrome f subunits of bc1 and b6f complexes are presented. Q-band excitation provides a particularly effective probe of the local heme environments of these species. The effects of protein conformation (particularly axial ligation) on heme structure and function were further investigated by comparison of spectra obtained from native subunits to those of a site directed c1 mutant (M183L) and various pH-dependent species of horse heart cytochrome c. In general, all species examined displayed variability in their axial amino acid ligation that suggests a good deal of flexibility in their hemepocket conformations. Surprisingly, the large scale protein rearrangements that accompany axial ligand replacement have little or no effect on macrocycle geometry in these species. This indicates the identity and/or conformation of the peptide linkage between the two cysteines that are covalently linked to the heme periphery may determine heme geometry.

Brassica↗
PubMed1999

Multiple types of spontaneous excitatory synaptic currents in salamander retinal ganglion cells.

Spontaneous and light-evoked excitatory postsynaptic currents (sEPSCs and leEPSCs) in retinal ganglion cells of the larval tiger salamander were recorded under voltage clamp conditions from living retinal slices. sEPSCs were isolated from the spontaneous inhibitory postsynaptic currents (sIPSCs) by application of 100 M picrotoxin+1 microM strychnine. In addition to the previously reported sEPSCs [K. Matsui, N. Hosoi, M. Tachibana, Excitatory synaptic transmission in the inner retina: pair recordings of bipolar cells and neurons of the ganglion cell layer, J. Neurosci. 18 (1998) 4500-4510; W.R. Taylor, E. Chen, D.R. Copenhagen, Characterization of spontaneous excitatory synaptic currents in salamander retinal ganglion cells, J. Physiol. 486 (1995) 207-221] [which are equivalent to our fast AMPA receptor-mediated sEPSCs (fAMPAsEPSCs)], we found another type of AMPA receptor-mediated sEPSC with slower rise and decay time courses and larger peak amplitudes (sAMPAsEPSCs), and the NMDA receptor-mediated sEPSCs (NMDAsEPSCs) in ON-OFF ganglion cells. The frequency of all three types of sEPSCs is greatly reduced by cobalt (with zero calcium) and increased by hyperosmotic solution, suggesting that these events are mediated by calcium-dependent exocytosis of glutamatergic synaptic vesicles. The amplitude histograms of sEPSCs do not show multiple peaks, suggesting that larger events are not discrete multiples of elementary events, or quanta, of similar neurotransmitter contents, as in the neuromuscular junction [P. Fatt, B. Katz, Spontaneous subthreshold activity at motor nerve endings, J. Physiol. 117 (1952) 109-128]. The average I-V relations of the fAMPAsEPSCs and sAMPAsEPSCs were outward rectified with reversal potentials at -12.2 mV and -10.8 mV, and that of the NMDAsEPSCs was N-shaped with a reversal potential at -5.8 mV. The average conductance increase associated with a single fAMPAsEPSC, a single sAMPAsEPSC, and a single NMDAsEPSC were 163. 26+/-51.02 pS, 233.33+/-163.64 pS, and 37.5+/-50.0 pS at -110 mV; 241.67+/-22.92 pS, 444.90+/-469.94 pS, and 25.93+/-70.37 pS at -60 mV; and 440.48+/-183.33 pS, 1,192.68+/-651.22 pS, and 517.71+/-238. 24 pS at +30 mV, respectively. The average frequency of the three sEPSCs at +30 mV were 15 Hz, 3.7 Hz and 3.6 Hz, respectively. The rise time (time to peak) of fAMPAsEPSCs was 1.5+/-1.05 ms and the decay time could be fitted with a single exponential with an average time constant of 3.4+/-4.1 ms. The rise and decay time course of the sAMPAsEPSCs and NMDAsEPSCs were much slower and sawtooth-shaped, and each 'sawtooth' had time course and amplitude similar to those of individual fAMPAsEPSCs. We propose that each fAMPAsEPSC is mediated by single or synchronized multiples of glutamatergic synaptic vesicles from bipolar cells, and each sAMPAsEPSC or NMDAsEPSC is mediated by larger clusters of synaptic vesicles triggered by spontaneous calcium spikes in bipolar cell axon terminals [J. Burrone, L. Lagnado, Electrical resonance and calcium influx in the synaptic terminal of depolarizing bipolar cells from the goldfish retina, J. Physiol. 505 (1997) 571-584; D. Zenisek, G. Matthews, Calcium action potentials in retinal bipolar neurons, Vis. Neurosci. 15 (1998) 69-75].

Action Potentials↗
PubMed1999

Origin of HIV-1 in the chimpanzee Pan troglodytes troglodytes.

The human AIDS viruses human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) represent cross-species (zoonotic) infections. Although the primate reservoir of HIV-2 has been clearly identified as the sooty mangabey (Cercocebus atys), the origin of HIV-1 remains uncertain. Viruses related to HIV-1 have been isolated from the common chimpanzee (Pan troglodytes), but only three such SIVcpz infections have been documented, one of which involved a virus so divergent that it might represent a different primate lentiviral lineage. In a search for the HIV-1 reservoir, we have now sequenced the genome of a new SIVcpzstrain (SIVcpzUS) and have determined, by mitochondrial DNA analysis, the subspecies identity of all known SIVcpz-infected chimpanzees. We find that two chimpanzee subspecies in Africa, the central P. t. troglodytes and the eastern P. t. schweinfurthii, harbour SIVcpz and that their respective viruses form two highly divergent (but subspecies-specific) phylogenetic lineages. All HIV-1 strains known to infect man, including HIV-1 groups M, N and O, are closely related to just one of these SIVcpz lineages, that found in P. t. troglodytes. Moreover, we find that HIV-1 group N is a mosaic of SIVcpzUS- and HIV-1-related sequences, indicating an ancestral recombination event in a chimpanzee host. These results, together with the observation that the natural range of P. t. troglodytes coincides uniquely with areas of HIV-1 group M, N and O endemicity, indicate that P. t. troglodytes is the primary reservoir for HIV-1 and has been the source of at least three independent introductions of SIVcpz into the human population.

Acquired Immunodeficiency Syndrome↗
PubMed1999

Retinal ganglion cell dysfunction induced by hypoxia and glutamate: potential neuroprotective effects of beta-blockers.

The objective of this study was to examine the effects of hypoxia, glutamate, and beta-blockers on the electrical activities of retinal ganglion cells. Single-unit extracellular and whole-cell voltage clamp recording techniques were used to record electrical activities from ganglion cells in the tiger salamander retina. This was performed under physiologic conditions, hypoxia, or elevated exogenous or endogenous glutamate levels. Light-evoked spike activities, glutamate-induced currents, and voltage-gated sodium and calcium currents were measured in the presence of the beta-1 selective antagonist betaxolol or the nonselective antagonist timolol. Hypoxia resulted in suppressing or blocking the OFF responses in the majority of ON-OFF ganglion cells tested, whereas the ON responses were only slightly affected. The presence of increased glutamate had similar findings and demonstrated an increase in the spontaneous firing rate of retinal ganglion cells. Betaxolol (2-50 microM) reduced the rate of spontaneous firing of retinal ganglion cells induced by glutamate. At 2 to 50 microM, betaxolol reversibly reduced the voltage-gated sodium currents and calcium currents in retinal ganglion cells. Timolol (up to 100 microM) did not demonstrate any detectable action on these currents. The physiologic responses of retinal ganglion cells to hypoxia or elevated glutamate levels in this animal model appear to be very similar. Although short-term exposure to hypoxia and glutamate used in this study exerts reversible actions on ganglion cells and does not induce permanent cell damage, such initial physiologic actions are likely to be precursors of permanent cell damage. Thus, hypoxia and elevated glutamate levels in the retina may represent a final pathway in diseases affecting retinal ganglion cells, such as glaucoma. Similar damage could result from different factors, such as decreased perfusion-induced ischemia or anomalous neuronal processing of glutamate. Betaxolol exerts its primary neuronal actions on retinal ganglion cells. It reversibly blocked voltage-gated calcium current and reduced the spontaneous firing rate by suppressing glutamate-gated currents and sodium currents in ganglion cells. These actions may protect ganglion cells from damage caused by ischemia or elevated glutamate levels.

Action Potentials↗
PubMed1999

Analysis of protein activity data by Gaussian stochastic process models.

The effects of certain chemical additives at maintaining a high level of activity in protein constructs during storage is investigated. We use a semiparametric regression technique to model the effects of the additives on protein activity. The model is extended to handle categorical explanatory variables. On the basis of the available data, the important factors are estimated to be buffer, detergent, protein concentration, and storage temperature. The relationships among protein activity and these factors appear to be moderately nonlinear with strong interaction effects. These features are revealed in a data-adaptive way by the semi parametric model, without explicit modeling of the nonlinearities or interactions. We use cross-validation to assess the fit of our model. The protein activity response appears to be extremely erratic. We recommend several sets of storage conditions and that further design points be chosen in regions around these estimated optima.

Data Interpretation, Statistical↗
PubMed1999

Time course of neurone-specific enolase and S-100 protein release during and after coronary artery bypass grafting.

Serum neurone-specific enolase (NSE) and S-100 protein are well established as markers of cerebral injury, and have been used as markers of neuronal and glial cell damage, respectively, after cardiac surgery with cardiopulmonary bypass (CPB), but the speed of their increase during CPB has not been studied. Therefore, we have investigated the time course of NSE and S-100 release during and after CPB. We studied 18 adult patients undergoing elective coronary artery bypass grafting (CABG). Standard hypothermic (32 degrees C) pulsatile bypass with membrane oxygenation was used. Blood samples were obtained at induction, before bypass, before rewarming, at the end of rewarming, 10 min, 1 h and 8 h after bypass and 1, 2 and 3 days after surgery. NSE and S-100 were assayed using immunoradiometric assay kits (Sangtec Medical). NSE and S-100 release followed similar time courses. Both increased sharply during bypass, reached peak concentrations at the end of rewarming (mean 25.55 (SEM 2.79) and 1.65 (0.23) microgram litre-1, respectively), had decreased significantly by the end of operation and returned to pre-bypass concentrations by the second day after surgery. No patient developed a major neurological deficit. When using NSE and S-100 assays to study cerebral dysfunction in relation to CPB, postoperative samples miss peak (end-bypass) concentrations, and studies should be designed to include intraoperative samples.

Adult↗
PubMed1999

Detection of phylogenetically diverse human immunodeficiency virus type 1 groups M and O from plasma by using highly sensitive and specific generic primers.

The high degree of genetic diversity within human immunodeficiency virus type 1 (HIV-1), which includes two major groups, M (major) and O (outlier), and various env subtypes within group M (subtypes A to J), has made designing assays that will detect all known HIV-1 strains difficult. We have developed a generic primer set based on the conserved immunodominant region of transmembrane protein gp41 that can reliably amplify as few as 10 copies/PCR of viral DNA from near-full-length clones representing group M subtypes A to H (subtypes I and J were not available). The assay is highly sensitive in detecting plasma viral RNA from HIV-1 strains of diverse geographic origins representing different subtypes of HIV-1 group M as well as HIV-1 group O. Of the 253 group M plasma specimens (subtypes A, 68 specimens; B, 71; C, 19; D, 27; E, 23; F, 33; and G, 12), 250 (98.8%) were amplified by using the gp41 M/O primer set. More importantly, all 32 (100%) group O plasma samples were also amplified with these primers. In vitro spiking experiments further revealed that the assay could reliably detect as few as 25 copies/ml of viral RNA and gave positive signals in HIV-1-seropositive specimens with plasma copy numbers below the limits of detection by all commercially available viral load assays. In addition, analysis of five seroconversion panels indicated that the assay is highly sensitive for early detection of plasma viremia during the "window period." Thus, the highly sensitive assay will be useful for early detection of HIV-1 in clinical specimens from all known HIV-1 infections, regardless of their genotypes and geographic origins.

Acquired Immunodeficiency Syndrome↗
PubMed1998

At least four loci and gender are associated with susceptibility to the chemical induction of lung adenomas in A/J x BALB/c mice.

Four putative quantitative trait loci (QTLs) that influence susceptibility to the induction of lung adenomas by urethane in an F2 cross between A/J and BALB/cOlaHsd have been mapped. Following microsatellite typing of mice with resistant and susceptible phenotypes at 97 microsatellite marker loci, a major locus was identified on chromosome 18 with a lod score of 15. This was responsible for an 8- to 10-fold increase in tumor multiplicity in males and females, respectively, having the AA and CC genotypes at the D18Mit188 marker locus. It mapped close to Dcc (deleted in colorectal cancer). A locus on chromosome 4 (lod score 6.5) had the resistant allele in strain A/J and the susceptible allele in BALB/c, with a 14-fold difference in tumor multiplicity between mice of the AA and CC genotypes. This mapped close to the Cdkn2a (cyclin-dependent kinase inhibitor 2A) locus, which is commonly deleted in mouse lung tumors. Two loci with smaller effects (lod scores 3.03 and 3.25) were identified on chromosomes 1 and 11. There was also significant sexual dimorphism in tumor multiplicity both among 151 F2 hybrids and among 52 mice resulting from a backcross to strain A/J, with males having higher tumor counts than females.

Adenoma↗
PubMed1998

Effects of mechano-gated cation channel blockers on Xenopus oocyte growth and development.

The putative role(s) of a mechanically gated (MG) cation channel in Xenopus oocyte growth, maturation, fertilization and embryogenesis has been examined. Using a pharmacological approach, we have tested the effects of the MG channel blockers, gadolinium, gentamicin and amiloride on the above developmental events. Our results indicate that oocyte maturation, fertilization and early embryogenesis (up to the free-swimming stage 45) can proceed normally in the presence of concentrations of agents that either completely abolish (i.e., > or = 10 microM Gd3+) or partially block (i.e., 1 mM gentamicin) single MG channel activity as measured by patch-clamp recording. However, we also find that higher concentrations of Gd3+ (> or = 50 microM) can lead to an increased percentage (> 20%) of axis-perturbed embryos compared with control (< 1%) and that amiloride (0.5 mM) reduces the success of fertilization (from 100% to < 50%) and increases mortality (by approximately 75%) in developing embryos. Furthermore, we find that all three agents inhibit oocyte growth in vitro. However, their order of effectiveness (amiloride > gentamicin > Gd3+) is opposite to their order for blocking MG channels (Gd3+ >> gentamicin > amiloride). These discrepancies indicated that the drugs effects occur by mechanisms other than, or in addition to, MG channel block. Our results provide no compelling evidence for the idea that MG channel activity is critical for development in Xenopus. This could mean that there are other mechanisms in the oocyte that can compensate when MG channel activity is blocked or that the protein that forms the channel can undergo additional interactions that result in a function insensitive to MG channel blockers.

Amiloride↗
PubMed1998

Q-band resonance Raman spectra of oxidized and reduced mitochondrial bc1 complexes.

Recently published crystallographic studies of mitochondrial bc1 complexes have stimulated renewed interest in the active site architecture of these important integral membrane proteins. We present resonance Raman spectra obtained via variable excitation within the heme Q-band from samples poised in several different net redox states. Appropriate subtraction and polarization analysis allows the vibrational behavior of the individual heme bL,bH, and c1 sites to be assessed. The spectra of the b hemes are particularly noteworthy. They exhibit evidence for a protonation equilibrium involving heme axial ligands and reveal a marked structural heterogeneity at the heme bH site that most likely involves nonplanar distortions of the macrocycle. The possible implications of these findings for heme functionality are discussed.

Animals↗
PubMed1998

Postsynaptic responses of horizontal cells in the tiger salamander retina are mediated by AMPA-preferring receptors.

The postsynaptic responses of sign-preserving second-order retinal neurons (horizontal cells (HCs) and off-bipolar cells) are mediated by CNQX-sensitive AMPA/KA glutamate receptors. In this study we used receptor-specific allosteric regulators of desensitization and selected antagonists to determine the glutamate receptor subtypes in tiger salamander horizontal cells. Two approaches were employed in this study. The first was to measure postsynaptic currents induced by exogenously applied glutamate under voltage clamp conditions in living retinal slices; and the second was to record voltage responses controlled by endogenous glutamate released from photoreceptors in whole retinas. Application of 100 microM cyclothiazide (a specific AMPA receptor desensitization blocker) enhanced the glutamate-induced current by about 5 fold. In contrast, 300 microgram ml-1 Co nA (a specific kainate receptor desensitization blocker), had no effect. GYKI 52466 (a specific AMPA receptor antagonist) at 30 microM almost completely suppressed the glutamate-induced inward current in HCs. Cyclothiazide at 100 microM depolarized the HC dark membrane potential by about 5 mV and reduced the amplitudes of the voltage responses to dim lights, but enhanced the voltage responses to bright lights. Cyclothiazide had no effect on either the dark potential or the light responses of rods and cones. Con A at 300 microgram ml-1 had no effect on either the dark potential or the light responses of the HC. GYKI 52466 (30 microM) hyperpolarized the HC dark membrane potential by about 55 mV and almost completely suppressed the light responses. We conclude from these results that the postsynaptic glutamate- and light-induced responses in the tiger salamander retinal horizontal cells are mediated by AMPA-preferring, and not kainate-preferring glutamate receptors. The functional roles of AMPA receptors and their desensitization kinetics in visual information processing are discussed.

Ambystoma↗
PubMed1998

Prolonged intercostal nerve blockade in sheep using controlled-release of bupivacaine and dexamethasone from polymer microspheres.

BACKGROUND: Previous work from the authors' group characterized a prolonged percutaneous blockade of the sciatic nerve in rats using bupivacaine-dexamethasone microspheres. The goals of the current study are to examine the (1) efficacy of bupivacaine microspheres with and without dexamethasone for intercostal blockade in sheep; (2) scaling of dose and duration with a 100-fold increase in body size from rats to sheep; (3) local toxicity and adverse systemic reactions to bupivacaine microspheres with and without dexamethasone. METHODS: Intercostal blocks were performed percutaneously in sedated sheep. Sensory blockade was measured at repeated time points by absent flinch response to skin pinch. Plasma bupivacaine concentrations were measured using high performance liquid chromatography. Chest wall specimens were examined by light microscopy. RESULTS: The duration of intercostal blockade increased with bupivacaine dose for animals receiving from 8 to 80 mg/kg of microspheres with and without dexamethasone. At each dose, microspheres containing dexamethasone had a longer duration of block than microspheres without dexamethasone. From 8 to 80 mg/kg, the mean duration of block with bupivacaine-dexamethasone microspheres increased from 4 to 13 days. Plasma concentrations of bupivacaine remained 10-fold below the convulsive EC50 concentration for sheep. Chest wall histology showed a significant granulomatous reaction around bupivacaine microspheres but not around bupivacaine-dexamethasone microspheres. CONCLUSIONS: A single administration of bupivacaine-dexamethasone microspheres produces an effective chest wall analgesia of several days' duration. This may prove useful clinically for thoracic surgery or trauma.

Anesthetics, Local↗
PubMed1998

Natural infection of a household pet red-capped mangabey (Cercocebus torquatus torquatus) with a new simian immunodeficiency virus.

A seroprevalence survey was conducted for simian immunodeficiency virus (SIV) antibody in household pet monkeys in Gabon. Twenty-nine monkeys representing seven species were analyzed. By using human immunodeficiency virus type 2 (HIV-2)/SIVsm, SIVmnd, and SIVagm antigens, one red-capped mangabey (RCM) (Cercocebus torquatus torquatus) was identified as harboring SIV-cross-reactive antibodies. A virus isolate, termed SIVrcm, was subsequently established from this seropositive RCM by cocultivation of its peripheral blood mononuclear cells (PBMC) with PBMC from seronegative humans or RCMs. SIVrcm was also isolated by cocultivation of CD8-depleted RCM PBMC with Molt 4 clone 8 cells but not with CEMx174 cells. The lack of growth in CEMx174 cells distinguished this new SIV from all previously reported sooty mangabey-derived viruses (SIVsm), which grow well in this cell line. SIVrcm was also successfully transmitted (cell free) to human and rhesus PBMC as well as to Molt 4 clone 8 cells. To determine the evolutionary origins of this newly identified virus, subgenomic pol (475 bp) and gag (954 bp) gene fragments were amplified from infected cell culture DNA and sequenced. The position of SIVrcm relative to those of members of the other primate lentivirus lineages was then examined in evolutionary trees constructed from deduced protein sequences. This analysis revealed significantly discordant phylogenetic positions of SIVrcm in the two genomic regions. In trees derived from partial gag sequences, SIVrcm clustered independently from all other HIV and SIV strains, consistent with a new primate lentivirus lineage. However, in trees derived from pol sequences, SIVrcm grouped with the HIV-1/SIVcpz lineage. These findings suggest that the SIVrcm genome is mosaic and possibly is the result of a recombination event involving divergent lentiviruses in the distant past. Further analysis of this and other SIVrcm isolates may shed new light on the origin of HIV-1.

Adaptation, Physiological↗
PubMed1998

An isolate of human immunodeficiency virus type 1 originally classified as subtype I represents a complex mosaic comprising three different group M subtypes (A, G, and I).

Full-length reference clones and sequences are currently available for eight human immunodeficiency virus type 1 (HIV-1) group M subtypes (A through H), but none have been reported for subtypes I and J, which have only been identified in a few individuals. Phylogenetic information for subtype I, in particular, is limited since only about 400 bp of env gene sequences have been determined for just two epidemiologically linked viruses infecting a couple who were heterosexual intravenous drug users from Cyprus. To characterize subtype I in greater detail, we employed long-range PCR to clone a full-length provirus (94CY032.3) from an isolate obtained from one of the individuals originally reported to be infected with this subtype. Phylogenetic analysis of C2-V3 env gene sequences confirmed that 94CY032.3 was closely related to sequences previously classified as subtype I. However, analysis of the remainder of its genome revealed various regions in which 94CY032.3 was significantly clustered with either subtype A or subtype G. Only sequences located in vpr and nef, as well as the middle portions of pol and env, formed independent lineages roughly equidistant from all other known subtypes. Since these latter regions most likely have a common origin, we classify them all as subtype I. These results thus indicate that the originally reported prototypic subtype I isolate 94CY032 represents a triple recombinant (A/G/I) with at least 11 points of recombination crossover. We also screened HIV-1 recombinants with regions of uncertain subtype assignment for the presence of subtype I sequences. This analysis revealed that two of the earliest mosaics from Africa, Z321B (A/G/?) and MAL (A/D/?), contain short segments of sequence which clustered closely with the subtype I domains of 94CY032.3. Since Z321 was isolated in 1976, subtype I as well as subtypes A and G must have existed in Central Africa prior to that date. The discovery of subtype I in HIV-1 hybrids from widely distant geographic locations also suggests a more widespread distribution of this virus subtype, or at least segments of it, than previously recognized.

Base Sequence↗