Signaling mediated by the closely related mammalian Rho family GTPases TC10 and Cdc42 suggests distinct functional pathways.

The mammalian Rho family GTPases TC10 and Cdc42 share many properties. Activated forms of both proteins stimulate transcription mediated by nuclear factor kappaB, serum response factor, and the cyclin D1 promoter; activate c-Jun NH2-terminal kinase; cooperate with activated Raf to transform NIH-3T3 cells; and, by a mechanism independent of all of these effects, induce filopodia formation. In contrast, previously reported differences between TC10 and Cdc42 are not striking. We now present studies of TC10 and Cdc42 in cell culture that reveal clear functional differences: (a) wild-type TC10 localizes predominantly to the plasma membrane and less extensively to a perinuclear membranous compartment, whereas wild-type Cdc42 localizes predominantly to this compartment and less extensively to the plasma membrane; (b) expression of Rho guanine nucleotide dissociation inhibitor alpha results in a redistribution of wild-type Cdc42 to the cytosol but has no effect on the plasma membrane localization of wild-type TC10; (c) TC10 fails to rescue a Saccharomyces cerevisiae cdc42 mutation, unlike mammalian Cdc42; (d) dominant negative Cdc42, but not dominant negative TC10, inhibits neurite outgrowth in PC12 cells stimulated by nerve growth factor; and (e) activation of nuclear factor kappaB-dependent transcription by Cdc42, but not by TC10, is inhibited by sodium salicylate. These findings point to distinct pathways in which TC10 and Cdc42 may act and distinct modes of regulation of these proteins.

Cellular functions of TC10, a Rho family GTPase: regulation of morphology, signal transduction and cell growth.

The small Ras-related GTPase, TC10, has been classified on the basis of sequence homology to be a member of the Rho family. This family, which includes the Rho, Rac and CDC42 subfamilies, has been shown to regulate a variety of apparently diverse cellular processes such as actin cytoskeletal organization, mitogen-activated protein kinase (MAPK) cascades, cell cycle progression and transformation. In order to begin a study of TC10 biological function, we expressed wild type and various mutant forms of this protein in mammalian cells and investigated both the intracellular localization of the expressed proteins and their abilities to stimulate known Rho family-associated processes. Wild type TC10 was located predominantly in the cell membrane (apparently in the same regions as actin filaments), GTPase defective (75L) and GTP-binding defective (31N) mutants were located predominantly in cytoplasmic perinuclear regions, and a deletion mutant lacking the carboxyl terminal residues required for post-translational prenylation was located predominantly in the nucleus. The GTPase defective (constitutively active) TC10 mutant: (1) stimulated the formation of long filopodia; (2) activated c-Jun amino terminal kinase (JNK); (3) activated serum response factor (SRF)-dependent transcription; (4) activated NF-kappaB-dependent transcription; and (5) synergized with an activated Raf-kinase (Raf-CAAX) to transform NIH3T3 cells. In addition, wild type TC10 function is required for full H-Ras transforming potential. We demonstrate that an intact effector domain and carboxyl terminal prenylation signal are required for proper TC10 function and that TC10 signals to at least two separable downstream target pathways. In addition, TC10 interacted with the actin-binding and filament-forming protein, profilin, in both a two-hybrid cDNA library screen, and an in vitro binding assay. Taken together, these data support a classification of TC10 as a member of the Rho family, and in particular, suggest that TC10 functions to regulate cellular signaling to the actin cytoskeleton and processes associated with cell growth.

Isolated mammalian and Schizosaccharomyces pombe ran-binding domains rescue S. pombe sbp1 (RanBP1) genomic mutants.

Mammalian Ran-binding protein-1 (RanBP1) and its fission yeast homologue, sbp1p, are cytosolic proteins that interact with the GTP-charged form of Ran GTPase through a conserved Ran-binding domain (RBD). In vitro, this interaction can accelerate the Ran GTPase-activating protein-mediated hydrolysis of GTP on Ran and the turnover of nuclear import and export complexes. To analyze RanBP1 function in vivo, we expressed exogenous RanBP1, sbp1p, and the RBD of each in mammalian cells, in wild-type fission yeast, and in yeast whose endogenous sbp1 gene was disrupted. Mammalian cells and wild-type yeast expressing moderate levels of each protein were viable and displayed normal nuclear protein import. sbp1(-) yeast were inviable but could be rescued by all four exogenous proteins. Two RBDs of the mammalian nucleoporin RanBP2 also rescued sbp1(-) yeast. In mammalian cells, wild-type yeast, and rescued mutant yeast, exogenous full-length RanBP1 and sbp1p localized predominantly to the cytosol, whereas exogenous RBDs localized predominantly to the cell nucleus. These results suggest that only the RBD of sbp1p is required for its function in fission yeast, and that this function may not require confinement of the RBD to the cytosol. The results also indicate that the polar amino-terminal portion of sbp1p mediates cytosolic localization of the protein in both yeast and mammalian cells.

A T42A Ran mutation: differential interactions with effectors and regulators, and defect in nuclear protein import.

Ran, the small, predominantly nuclear GTPase, has been implicated in the regulation of a variety of cellular processes including cell cycle progression, nuclear-cytoplasmic trafficking of RNA and protein, nuclear structure, and DNA synthesis. It is not known whether Ran functions directly in each process or whether many of its roles may be secondary to a direct role in only one, for example, nuclear protein import. To identify biochemical links between Ran and its functional target(s), we have generated and examined the properties of a putative Ran effector mutation, T42A-Ran. T42A-Ran binds guanine nucleotides as well as wild-type Ran and responds as well as wild-type Ran to GTP or GDP exchange stimulated by the Ran-specific guanine nucleotide exchange factor, RCC1. T42A-Ran.GDP also retains the ability to bind p10/NTF2, a component of the nuclear import pathway. In contrast to wild-type Ran, T42A-Ran.GTP binds very weakly or not detectably to three proposed Ran effectors, Ran-binding protein 1 (RanBP1), Ran-binding protein 2 (RanBP2, a nucleoporin), and karyopherin beta (a component of the nuclear protein import pathway), and is not stimulated to hydrolyze bound GTP by Ran GTPase-activating protein, RanGAP1. Also in contrast to wild-type Ran, T42A-Ran does not stimulate nuclear protein import in a digitonin permeabilized cell assay and also inhibits wild-type Ran function in this system. However, the T42A mutation does not block the docking of karyophilic substrates at the nuclear pore. These properties of T42A-Ran are consistent with its classification as an effector mutant and define the exposed region of Ran containing the mutation as a probable effector loop.

Involvement of Sonic hedgehog (Shh) in mouse embryonic lung growth and morphogenesis.

Branching morphogenesis of the embryonic lung requires interactions between the epithelium and the mesenchyme. Previously, we reported that Sonic hedgehog (Shh) transcripts are present in the epithelium of the developing mouse lung, with highest levels in the terminal buds. Here, we report that transcripts of mouse patched (Ptc), the homologue of a Drosophila gene encoding a putative transmembrane protein required for hedgehog signaling, are expressed at high levels in the mesenchyme adjacent to the end buds. To investigate the function of SHH in lung development, Shh was overexpressed throughout the distal epithelium, using the surfactant protein-C (SP-C)-enhancer/promoter. Beginning around 16.5 dpc, when Shh and Ptc RNA levels are normally both declining, this treatment caused an increase in the ratio of interstitial mesenchyme to epithelial tubules in transgenic compared to normal lungs. Transgenic newborn mice die soon after birth. Histological analysis of the lungs at the light and electron microscope level shows an abundance of mesenchyme and the absence of typical alveoli. In vivo BrdU labeling indicates that Shh overexpression results in increased mesenchymal and epithelial cell proliferation at 16.5 and 17.5 dpc. However, analysis of CC-10 and SP-C expression reveals no significant inhibition in the differentiation of proximal and distal epithelial cells. The expression of genes potentially regulated by SHH was also examined. No difference could be observed between transgenic and control lungs in either the level or distribution of Bmp4, Wnt2 and Fgf7 RNA. By contrast, Ptc is clearly upregulated in the transgenic lung. These results thus establish a role for SHH in lung morphogenesis, and suggest that SHH normally regulates lung mesenchymal cell proliferation in vivo.

The small nuclear GTPase Ran: how much does it run?

Ran is one of the most abundant and best conserved of the small GTP binding and hydrolyzing proteins of eukaryotes. It is located predominantly in cell nuclei. Ran is a member of the Ras family of GTPases, which includes the Ras and Ras-like proteins that regulate cell growth and division, the Rho and Rac proteins that regulate cytoskeletal organization and the Rab proteins that regulate vesicular sorting. Ran differs most obviously from other members of the Ras family in both its nuclear localization, and its lack of sites required for post-translational lipid modification. Ran is, however, similar to other Ras family members in requiring a specific guanine nucleotide exchange factor (GEF) and a specific GTPase activating protein (GAP) as stimulators of overall GTPase activity. In this review, the multiple cellular functions of Ran are evaluated with respect to its known biochemistry and molecular interactions.

Separate domains of the Ran GTPase interact with different factors to regulate nuclear protein import and RNA processing.

The small Ras-related GTP binding and hydrolyzing protein Ran has been implicated in a variety of processes, including cell cycle progression, DNA synthesis, RNA processing, and nuclear-cytosolic trafficking of both RNA and proteins. Like other small GTPases, Ran appears to function as a switch: Ran-GTP and Ran-GDP levels are regulated both by guanine nucleotide exchange factors and GTPase activating proteins, and Ran-GTP and Ran-GDP interact differentially with one or more effectors. One such putative effector, Ran-binding protein 1 (RanBP1), interacts selectively with Ran-GTP. Ran proteins contain a diagnostic short, acidic, carboxyl-terminal domain, DEDDDL, which, at least in the case of human Ran, is required for its role in cell cycle regulation. We show here that this domain is required for the interaction between Ran and RanBP1 but not for the interaction between Ran and a Ran guanine nucleotide exchange factor or between Ran and a Ran GTPase activating protein. In addition, Ran lacking this carboxyl-terminal domain functions normally in an in vitro nuclear protein import assay. We also show that RanBP1 interacts with the mammalian homolog of yeast protein RNA1, a protein involved in RNA transport and processing. These results are consistent with the hypothesis that Ran functions directly in at least two pathways, one, dependent on RanBP1, that affects cell cycle progression and RNA export, and another, independent of RanBP1, that affects nuclear protein import.

Sequential evaluation of plasma retinol-binding protein response to vitamin A administration in very-low-birth-weight neonates.

Vitamin A (retinol) deficiency is associated with impaired healing from lung injury in very-low-birth-weight (VLBW) neonates susceptible to bronchopulmonary dysplasia (BPD). Vitamin A supplementation from birth may ameliorate this adverse outcome. We hypothesized that plasma retinol-binding protein (RBP) response to vitamin A administration, which provides a dynamic measure to vitamin A status, might be useful for early recognition of vitamin A deficiency in VLBW neonates at risk for BPD. We prospectively studied 20 VLBW neonates (inclusion criteria: birth weight < 1300 g, gestational age < 30 weeks, need for supplemental oxygen and mechanical ventilation for > 24 h after birth) who were eligible to receive vitamin A supplementation. In addition to sequential assessment of vitamin A status, we measured plasma RBP just before and 3 and 6 h after an intramuscular injection of vitamin A (2000 IU/kg retinyl palmitate) on Postnatal Days 1, 7, 15, 21, 29, and 43. The percentage increase in plasma RBP (delta-RBP) was calculated. A high plasma delta-RBP value ( > 8%) is indicative of vitamin A deficiency. Based on pulmonary outcome, the infants were divided into two groups: BPD (n = 12) and No BPD (n = 8). Mean vitamin A intake ranged from 1414 to 2114 IU/kg/day and did not differ between infant groups. Mean plasma vitamin A concentration increased from baseline levels on Postnatal Day 1 to levels within the desired range of 1.05-2.10 mumol/liter (30.0-60.0 micrograms/dl) during supplementation period in both infant groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue-specific expression of Ran isoforms in the mouse.

Ran genes encode a family of well-conserve small nuclear GTPases (Ras-related nuclear proteins), whose function is implicated in both normal cell cycle progression and the transport of RNA and proteins between the nucleus and the cytoplasm. Previous studies of Ran proteins have utilized cell-free systems, yeasts, and cultured mammalian cells. We have now characterized patterns of Ran gene expression in the mouse. Serum starvation suppressed Ran gene transcription in mouse 3T3 cells. Ran mRNA reappeared in cells within 3 h after refeeding. A single Ran mRNA species was detected at low levels in most somatic tissues of the adult mouse. In testis, this Ran mRNA was abundant, as were other larger transcripts. Analysis of testis-derived Ran cDNA clones revealed the presence of two transcripts, one specifying an amino acid sequence identical to that of human Ran/TC4 and one specifying an amino acid sequence 94% identical. Northern blotting and reverse transcriptase-PCR assays with oligonucleotide probes and primers specific for each transcript demonstrated that the isoform identical to Ran/TC4 was expressed in both somatic tissues and testis, while the variant form was transcribed only in testis. The existence of tissue-specific Ran isoforms may help to rationalize the diverse roles suggested for Ran by previous biochemical studies.

Intramuscular versus enteral vitamin A supplementation in very low birth weight neonates.

We conducted a randomized trial in very low birth weight neonates (n = 51) to determine whether vitamin A supplementation by enteral administration would increase plasma vitamin A concentrations to the same degree as by intramuscular administration. Mean plasma vitamin A concentrations were significantly higher in the intramuscular-administration group than in the enteral-administration group by postnatal day 7; this effect persisted throughout the remainder of the trial. At the dosage used in this trial, vitamin A supplementation by the enteral route is not as effective as that by the intramuscular route in very low birth weight neonates.

Aberrant function of the Ras-related protein TC21/R-Ras2 triggers malignant transformation.

Although the human Ras proteins are members of a large superfamily of Ras-related proteins, to date, only the proteins encoded by the three mammalian ras genes have been found to possess oncogenic potential. Among the known Ras-related proteins, TC21/R-Ras2 exhibits the most significant amino acid identity (55%) to Ras proteins. We have generated mutant forms of TC21 that possess amino acid substitutions analogous to those that activate Ras oncogenic potential [designated TC21(22V) and TC21(71L)] and compared the biological properties of TC21 with those of Ras proteins in NIH 3T3 and Rat-1 transformation assays. Whereas wild-type TC21 did not show any transforming potential in vitro, both TC21(22V) and TC21(71L) displayed surprisingly potent transforming activities that were comparable to the strong transforming activity of oncogenic Ras proteins. Like Ras-transformed cells, NIH 3T3 cells expressing mutant TC21 proteins formed foci of morphologically transformed cells in monolayer cultures, proliferated in low serum, formed colonies in soft agar, and developed progressive tumors in nude mice. Thus, TC21 is the first Ras-related protein to exhibit potent transforming activity equivalent to that of Ras. Furthermore, mutant TC21 proteins also stimulated constitutive activation of mitogen-activated protein kinases as well as transcriptional activation from Ras-responsive promoter elements (Ets/AP-1 and NF-kappa B). We conclude that aberrant TC21 function may trigger cellular transformation via a signal transduction pathway similar to that of oncogenic Ras and suggest that deregulated TC21 activity may contribute significantly to human oncogenesis.

Effects of mutant Ran/TC4 proteins on cell cycle progression.

Ran/TC4, a member of the RAS gene superfamily, encodes an abundant nuclear protein that binds and hydrolyzes GTP. Transient expression of a Ran/TC4 mutant protein deficient in GTP hydrolysis blocked DNA replication, suggesting a role for Ran/TC4 in the regulation of cell cycle progression. To test this possibility, we exploited an efficient transfection system, involving the introduction of cDNAs in the pMT2 vector into 293/Tag cells, to analyze phenotypes associated with mutant and wild-type Ran/TC4 expression. Expression of a Ran/TC4 mutant protein deficient in GTP hydrolysis inhibited proliferation of transfected cells by arresting them predominantly in the G2, but also in the G1, phase of the cell cycle. Deletion of an acidic carboxy-terminal hexapeptide from the Ran/TC4 mutant did not alter its nuclear localization but did block its inhibitory effect on cell cycle progression. These data suggest that normal progression of the cell cycle is coupled to the operation of a Ran/TC4 GTPase cycle. Mediators of this coupling are likely to include the nuclear regulator of chromosome condensation 1 protein and the mitosis-promoting factor complex.

Characterization of proteins that interact with the cell-cycle regulatory protein Ran/TC4.

The human Ras-related nuclear protein Ran/TC4 (refs 1-4) is the prototype of a well conserved family of GTPases that can regulate both cell-cycle progression and messenger RNA transport. Ran has been proposed to undergo tightly controlled cycles of GTP binding and hydrolysis, to operate as a GTPase switch whose GTP- and GDP-bound forms interact differentially with regulators and effectors. One known regulator, the protein RCC1 (refs 12, 13), interacts with Ran to catalyse guanine nucleotide exchange, and both RCC1 and Ran are components of an intrinsic checkpoint control that prevents the premature initiation of mitosis. To test and extend the GTPase-switch model, we searched for a Ran-specific GTPase-activating protein (GAP), and for putative effectors (proteins that interact specifically with Ran/TC4-GTP). We report here the identification of a Ran GAP and its use to characterize the GTP-hydrolysing properties of mutant Ran proteins, and the identification and cloning of a binding protein specific for Ran/TC4-GTP.

Ran/TC4: a small nuclear GTP-binding protein that regulates DNA synthesis.

Ran/TC4, first identified as a well-conserved gene distantly related to H-RAS, encodes a protein which has recently been shown in yeast and mammalian systems to interact with RCC1, a protein whose function is required for the normal coupling of the completion of DNA synthesis and the initiation of mitosis. Here, we present data indicating that the nuclear localization of Ran/TC4 requires the presence of RCC1. Transient expression of a Ran/TC4 protein with mutations expected to perturb GTP hydrolysis disrupts host cell DNA synthesis. These results suggest that Ran/TC4 and RCC1 are components of a GTPase switch that monitors the progress of DNA synthesis and couples the completion of DNA synthesis to the onset of mitosis.

Current therapy of bronchopulmonary dysplasia.

In summary, little progress has been made in the past several years with respect to the treatment of the baby with BPD. The conduct of convincing clinical research seems to be a casualty of budget cuts and a rush to learn the tools of molecular biology. To date, there are no clinical trials that have convincingly demonstrated that long-term diuretic, bronchodilator, vasodilator, or antioxidant therapy is effective in the treatment of chronic BPD. Short-term corticosteroid therapy hastens extubation, but long-term outcome is unaffected and serious questions remain about its safety. Multicenter clinical trials should be carefully designed and implemented to address the values of these therapies. In the design of these trials, care should be taken to stratify treatment groups for known risk factors for BPD. What are the future directions for the treatment of BPD? It is hoped that new BPD treatment strategies will be based on an improved understanding of mechanisms of lung repair and inflammation. Enzyme, gene, cytokine, antioxidant, and antiprotease therapies are being developed in animal models of lung injury. In addition, the use of lung transplantation has begun to be explored for severe cases of BPD. It is also possible, as has occurred in many chronic idiopathic diseases, that nonspecific treatment may prove beneficial. Perhaps it is only a matter of time before intravenous immunoglobulin, cyclosporine, methotrexate, or "biological response modifiers" will be administered to infants with severe BPD. For example, there is anecdotal evidence that recombinant human growth hormone may improve respiratory muscle function in adults with chronic obstructive pulmonary diseases. In the absence of convincing clinical trials, the clinician should reserve existing therapies for the ventilator-dependent infant or infants whose high oxygen requirement is prohibiting discharge or resulting in complex home care or frequent rehospitalizations. It should be emphasized that continuous oxygen therapy combined with avoidance of environmental inhalant and infectious hazards have the strongest rationale and widest margin of safety for treatment of the infant with BPD. Ironically, oxygen therapy is frequently underutilized and discontinued too rapidly. Early discontinuation of oxygen therapy with alveolar hypoxia results in feeding difficulty, slow growth, nutrient malabsorption, bronchoconstriction, and pulmonary hypertension. Oxygen therapy, although more cumbersome and certainly less glamorous than other pharmacologic agents, remains the essential element of BPD care.

HPV-16-related DNA sequences in Kaposi's sarcoma.

In the USA, Kaposi's sarcoma associated with the acquired immunodeficiency syndrome (AIDS-KS) is ten times more common in homosexual or bisexual men than in heterosexual men with AIDS. One explanation for this finding is that AIDS-KS may be caused by an infectious agent. Because there is a high incidence of human papillomavirus (HPV) infection, especially HPV-16, in homosexual men, we have sought HPV DNA sequences in Kaposi's sarcoma. We used the polymerase chain reaction with a primer pair specific for the highly conserved E6 region of HPV-16 to detect HPV-16 homologous DNA fragments in tumour tissues from 97 patients with KS and in KS-derived cell cultures. HPV DNA sequences were found in 11 of 69 KS skin tumours from homosexual men with AIDS-KS, in 3 of 11 KS biopsy specimens from homosexual men who had no clinical or laboratory evidence of HIV-infection, and in 5 of 17 KS skin lesions from HIV-1-negative elderly men and women with classic KS. The same primer pair amplified HPV-16 homologous fragments from two different continuous cell cultures derived from pleural effusion fluid of patients with pulmonary AIDS-KS and two continuous cell cultures derived from KS skin lesions. The findings suggest that HPV-16-related DNA sequences are associated with different forms of KS and may have a role in the pathogenesis of this neoplasm.

Detection of human T-lymphotropic virus-like particles in cultures of peripheral blood lymphocytes from patients with mycosis fungoides.

Because of seronegativity and absence of a leukemic phase in most patients with mycosis fungoides, a role for the human T-lymphotropic virus type I (HTLV-I) in this disease has remained tenuous. Virus particles are not seen in fresh isolates of skin or blood lymphocytes and the malignant cells (Sézary cells) have been difficult to culture. The availability of growth factors and biomolecular techniques have prompted a renewed attempt to find evidence of virus infection in these patients. We report here the successful culture of blood lymphocytes of 17 patients with mycosis fungoides and 1 patient with the Sézary syndrome. The cells of 2 additional patients failed to grow after 4-6 weeks in vitro. Ultrastructural analysis of the cultures showed an abundance of HTLV-like particles in the specimens of 18 of the 20 patients. Preliminary immunohistochemical studies carried out with various antisera directed against HTLV-I and the polymerase chain reaction utilizing a probe for a conserved region of the pol gene of HTLV-I were positive on only a portion of the specimens. Although definitive characterization of this organism awaits further analysis, it seems likely that circulating lymphocytes of all patients with mycosis fungoides harbor a virus that morphologically resembles HTLV-I.

Opposing effects of retinoic acid and dexamethasone on cellular retinol-binding protein ribonucleic acid levels in the rat.

Cellular retinol-binding protein (CRBP) is a potential mediator of vitamin A action. To determine whether retinoic acid and dexamethasone administration, alone and in combination, influence CRBP gene expression, adult female vitamin A-sufficient Sprague-Dawley rats randomly received 1) all-trans retinoic acid (100 micrograms) by intragastric intubation, 2) dexamethasone (2 micrograms/g BW) by ip injection, or 3) both all-trans retinoic acid and dexamethasone in the same doses. Control animals received either cottonseed oil by intragastric intubation or saline by ip injection. Six hours after treatment, lung and liver tissue were collected for Northern blot analysis with the radiolabeled cDNA specific for rat CRBP. Retinoic acid administration increased the amount of lung CRBP mRNA only, whereas dexamethasone decreased both lung and liver CRBP mRNA abundance. In animals treated with both retinoic acid and dexamethasone, CRBP mRNA abundance was also reduced. We conclude that CRBP gene expression can be modulated by both retinoic acid and dexamethasone in the vitamin A-sufficient animal. In the whole animal, our findings indicate that dexamethasone not only represses CRBP gene expression, but also opposes the effect of retinoic acid.

Evolutionary implications of primate endogenous retroviruses.

Endogenous DNA sequences related to retroviruses are probably present in all primates. By using approaches based on the polymerase chain reaction, two separate studies have revealed the evolutionary history of some of these sequences. In the first study, a retrovirus-like reverse transcriptase (RT) sequence homologous to that of Baboon endogenous virus (BaEV) has been identified in both Old World monkeys and African apes, but not in humans or Asian apes. This RT sequence is highly conserved at the amino acid level, but not the nucleotide level, in the baboon, African green monkey, Java macaque, chimpanzee, and gorilla. The patterns of nucleotide substitution indicate functional conservation and suggest that this RT sequence was present in the primate germline before apes and Old World monkeys diverged about 30 million years ago. In the second study, a comparison of endogenous proviral DNAs and their adjacent sequences has been used to analyze the evolutionary history of three previously reported human endogenous retroviruses, HERV-E(4.14), HERV-R(3), and HERV-Ia. It is shown that these retroviruses have also been resident in the primate line since before the ape-Old World monkey divergence. The implications of the presence of functionally conserved RT genes in the germlines of primates, and the potential for using integration sites as tools for analyzing phylogenetic relationships among primates and their retroviruses, are discussed.