Phototaxis signaling by the membrane complex of archaeal sensory rhodopsins and their transducers

Sensory rhodopsins I and II (SRI and SRII) are visual pigment-like phototaxis receptors in the archaeon Halobacterium salinarum. The receptor proteins each consist of a single polypeptide that folds into 7 $\alpha$-helical membrane-spanning segments forming an internal pocket where the chromophore retinal is bound. They transmit signals to their tightly bound transducer proteins, HtrI and HtrII, respectively, which in turn control a phosphotransfer pathway modulating the flagellar motors. SRI-HtrI mediates attractant responses to orange-light and repellent responses to UV light, while SRII-HtrII mediates repellent response to blue light. Experiments were designed to analyze the molecular processes in the SR-Htr complexes responsible for receptor activation, which previously had been shown by our laboratory to involve proton transfer reactions of the retinylidene Schiff base in the photoactive site, transfer of signals from receptor to transducer, and signaling specificity by the receptor-transducer complex.^ Site-directed mutagenesis and laser-flash kinetic spectroscopy revealed that His-166 in SRI (i) plays a role in the proton transfers both to and from the Schiffbase, either as a structurally critical residue or possibly as a direct participant, (ii) is involved in the modulation of SIU photoreaction kinetics by HtrI, and (iii) modulates the pKa of Asp-76, an important residue in the photoactive site, through a long-distance electrostatic interaction. Computerized cell tracking and motion analysis demonstrated that (iv) His-166 is crucial in phototaxis signaling: a spectrum of substitutions either eliminate signaling or greatly perturb the activation process that produces attractant and repellent signaling states of the receptor.^ The signaling states of SRI are communicated to HtrI, whose oligomeric structure and conformational changes were investigated by engineered sulfhydryl probes. It was found that signaling by the SRI-HtrI complex involves reversible conformational changes within a preexisting HtrI dimer, which is likely accomplished through a slight winding or unwinding of the two HtrT monomers via their loose coiled coil association. To elucidate which domains of the Htr dimers confer specificity for interaction with SRI or SRII, chimeras of HtrI and HtrII were constructed. The only determinant needed for functional and specific interaction with SRI or SRII was found to be the four transmembrane segments of the HtrI or HtrII dimers, respectively. The entire cytoplasmic parts of HtrI and HtrII, which include the functionally important signaling and adaptation domains, were interchangeable.^ These observations support a model in which SRI and SRII undergo conformational changes coupled to light-induced proton transfers in their photoactive sites, and that lateral helix-helix interactions with their cognate transducers' 4-helix bundle in the membrane relay these conformational changes into different states of the Htr proteins which regulate the down-stream phosphotransfer pathway. ^

Studies of subcellular localization and complex formation of the Agrobacterium tumefaciens transport ATPase VirB11

The VirB11 ATPase is an essential component of an Agrobacterium tumefaciens type IV bacterial secretion system that transfers oncogenic nucleoprotein complexes to susceptible plant cells. This dissertation investigates the subcellular localization and homo-oligomeric state of the VirB11 ATPase in order to provide insights about the assembly of the protein as a subunit of this membrane-associated transfer system. Subcellular fractionation studies and quantitative immunoblot analysis demonstrated that $\sim$30% of VirB11 partitioned as soluble protein and $\sim$70% was tightly associated with the bacterial cytoplasmic membrane. No differences were detected in VirB11 subcellular localization and membrane association in the presence or absence of other transport system components. Mutations in virB11 affecting protein function were mapped near the amino terminus, just upstream of a region encoding a Walker 'A' nucleotide-binding site, and within the Walker 'A' motif partitioned almost exclusively with the cytoplasmic membrane, suggesting that an activity associated with nucleotide binding could modulate the affinity of VirB11 for the cytoplasmic membrane. Merodiploid analysis of VirB11 mutant and truncation derivatives provided strong evidence that VirB11 functions as a homo- or heteromultimer and that the C-terminal half of VirB11 contains a protein interaction domain. A combination of biochemical and molecular genetic approaches suggested that VirB11 and the green fluorescence protein (GFP) formed a mixed multimer as demonstrated by immunoprecipitation experiments with anti-GFP antibodies. Second, a hybrid protein composed of VirB11 fused to the N-terminal DNA-binding domain of bacteriophage $\lambda$ cI repressor conferred immunity to $\lambda$ superinfection, demonstrating that VirB11 self-association promotes dimerization of the chimeric repressor. A conserved Walker 'A' motif, though required for VirB11 function in T-complex export, was not necessary for VirB11 self-association. Sequences in both the N- and the C-terminal halves of the protein were found to contribute to self-association of the full length protein. Chemical cross-linking experiments with His$\sb6$ tagged VirB11 suggested that VirB11 probably assembles into a higher order homo-oligomeric complex. ^

The effects of population density on the spread of disease

The objective of this study is to identify the relationship between population density and the initial stages of the spread of disease in a local population. This study proposes to concentrate on the question of how population density affects the distribution of the susceptible individuals in a local population and thus affects the spread of the disease, measles. Population density is measured by the average of the number of contacts with susceptible individuals by each individual in the population during a fixed-length time period. The term “contact with susceptible individuals” means sufficient contact between two people for the disease to pass from an infectious person to a susceptible person. The fixed-length time period is taken to be the average length of time an infected person is infectious without symptoms of the disease. For this study of measles, the time period will be seven days. ^ While much attention has been given to modeling the entire epidemic process of measles, attempts have not been made to study the characteristics of contact rates required to initiate an epidemic. This study explores the relationship between population density, given a specific herd immunity rate in the population, and initial rate of the spread of the disease by considering the underlying distribution of contacts with susceptibles by the individuals in the population. ^ This study does not seek to model an entire measles epidemic, but to model the above stated relationship for the local population within which the first infective person is introduced. This study describes the mathematical relationship between population density parameters and contact distribution parameters. ^ The results are displayed in graphs that show the effects of different population densities on the spread of disease. The results support the idea that the number of new infectives is strongly related to the distribution of susceptible contacts. The results also show large differences in the epidemic measures between populations with densities equal to four versus three. ^

Detection of enterotoxigenic Escherichia coli in foods by polymerase chain reaction

Enterotoxigenic Escherichia coli (ETEC) causes significant morbidity and mortality in infants of developing countries and is the most common cause of diarrhea in travelers to these areas. Enterotoxigenic Escherichia coli infections are commonly caused by ingestion of fecally contaminated food. A timely method for the detection of ETEC in foods would be important in the prevention of this disease. A multiplex polymerase chain reaction (PCR) assay which has been successful in detecting the heat-labile and heat-stable toxins of ETEC in stool was examined to determine its utility in foods. This PCR assay, preceded by a glass matrix and chaotropic DNA extraction, was effective in detecting high numbers of ETEC in a variety of foods. Ninety percent of 121 spiked food samples yielded positive results. Samples of salsa from Guadalajara, Mexico and Houston, Texas were collected and underwent DNA extraction and PCR. All samples yielded negative results for both the heat-labile and heat-stable toxins. Samples were also subjected to oligonucleotide probe analysis and resulted in 5 samples positive for ETEC. Upon dilution testing, it was found that positive PCR results only occurred when 12,000 to 1,000,000 bacteria were present in 200 mg of food. Although the DNA extraction and PCR method has been shown to be both sensitive and specific in stool, similar results were not obtained in food samples. ^

Immunology and HIV expression in skin, in vitro, and in response to ultraviolet light

HIV can enter the body through Langerhans cells, dendritic cells, and macrophages in skin mucosa, and spreads by lysis or by syncytia. Since UVL induces of HIV-LTR in transgenic mice mid in cell lines in vitro, we hypothesized that UVB may affect HIV in people and may affect HIV in T cells in relation to dose, apoptosis, and cytokine expression. To determine whether HIV is induced by UVL in humans, a clinical study of HIV+ patients with psoriasis or pruritus was conducted during six weeks of UVB phototherapy, Controls were HIV-psoriasis patients receiving UVB and HIV+ KS subjects without UVB.Blood and skin biopsy specimens were collected at baseline, weeks 2 and 6, and 4 weeks after UVL. AIDS-related skin diseases showed unique cytokine profiles in skin and serum at baseline. In patients and controls on phototherapy, we observed the following: (1) CD4+ and CD8+ T cell numbers are not significantly altered during phototherapy, (2) p24 antigen levels, and also HIV plasma levels increase in patients not on antiviral therapy, (3) HIV-RNA levels in serum or plasma. (viral load) can either increase or decrease depending on the patient's initial viral load, presence of antivirals, and skin type, (4) HIV-RNA levels in the periphery are inversely correlated to serum IL-10 and (5) HIV+ cell in skin increase after UVL at 2 weeks by RT-PCR in situ hybridization mid we negatively correlated with peripheral load. To understand the mechanisms of UVB mediated HIV transcription, we treated Jurkat T cell lines stably transfected with an HIV-LTR-luciferase plasmid only or additionally with tat-SV-40 early promoter with UVB (2 J/m2 to 200 J/m2), 50 to 200 ng/ml rhIL-10, and 10 μg/ml PHA as control. HIV promoter activity was measured by luciferase normalized to protein. Time points up to 72 hours were analyzed for HIV-LTR activation. HIV-LTR activation had the following properties: (1) requires the presence of Tat, (2) occurs at 24 hours, and (3) is UVB dose dependent. Changes in viability by MTS (3-(4,5-dimethyhhiazol-2-y1)-5-(3-carboxymethoxyphonyl)-2-(4-sulfophenyl)-2H-tetrazolium) mixed with PMS (phenazine methosulfate) solution and apoptosis by propidium iodide and annexin V using flow cytometry (FC) were seen in irradiated Jurkat cells. We determined that (1) rhIL-10 moderately decreased HIV-LTR activation if given before radiation and greatly decreases it when given after UVB, (2) HIV-LTR activation was low at doses of greater than 70 J/m2, compared to activation at 50 J/m2. (Abstract shortened by UMI.)^

The role of the amino terminus of Bacillus subtilis sigmaK in transcription initiation

The sigma (σ) subunit of eubacterial RNA polymerase (RNAP) is required for specific recognition of promoter DNA sequences and transcription initiation. Regulation of bacterial gene expression can be achieved by modulating a factor activity. The Bacillus subtilis sporulation a σ factor, σ K, controls gene expression of the late sporulation regulon. σ K is synthesized as an inactive precursor protein, pro-σ K, with a 20 amino acid pro sequence. Proteolytic processing of the pro sequence produces the active form, σK, which is able to bind to the core subunits of RNAP to direct gene expression. Thus, the pro sequence renders σK inactive in vivo. After processing, the amino terminus of σK consists of region 1.2, which is conserved among various σ factors. To understand the role of the amino terminus of σK, namely the pro sequence and region 1.2, mutagenesis of both regions was pursued. NH 2-terminal truncations of pro-σK were constructed to address how the pro sequence silences σK activity. The work described here shows that the pro sequence inhibits the ability of σ K to associate with the core subunits and that a deletion of only six amino acids of the pro sequence is sufficient to activate pro-σ K for DNA binding and transcription initiation to levels similar to σ K. Additionally, site directed mutagenesis was used to obtain single amino acid substitutions in region 1.2 to address the role of region 1.2 in σ K transcriptional activity. Two mutations were isolated, converting a lysine (K) to an alanine (A) at position three, and an asparagine (N) to a tyrosine (Y) at position five, both of which alter the efficiency of transcription initiation by RNAP containing the mutant σKs. Surprisingly, σ KK3A increased transcript production when compared to wild type. This increase is due to improvement in DNA affinity and increased stability of RNAP-DNA promoter open complexes. σKN5Y showed a decrease in transcription activity that is related to defects in the ability of RNAP to make the transition from the closed to open RNAP-DNA complex. Results of both the pro sequence and region 1.2 analyses indicate that the amino terminus of σK is important for transcription activity and this work adds to the increasing body of evidence that the amino termini of many σ factors modulate transcription initiation by RNAP. ^

Relationship between enterotoxigenic Escherichia coli and travelers' diarrhea in Mexico, from 1992 to 1997

The purpose of this study was to examine the relationship between enterotoxigenic ETEC and travelers' diarrhea over a period of five years in Guadalajara, Mexico. Specifically, this study identified and characterized ETEC from travelers with diarrhea. The objectives were to study the colonization factor antigens, toxins and antibiotic sensitivity patterns in ETEC from 1992 to 1997 and to study the molecular epidemiology of ETEC by plasmid content and DNA restriction fragment patterns. ^ In this survey of travelers' diarrhea in Guadalajara, Mexico, 928 travelers with diarrhea were screened for enteric pathogens between 1992 and 1997. ETEC were isolated in 195 (19.9%) of the patients, representing the most frequent enteric pathogen identified. ^ A total of 31 antimicrobial susceptibility patterns were identified among ETEC isolates over the five-year period. ^ The 195 ETEC isolates contained two to six plasmids each, which ranged in size from 2.0 to 23 kbp. ^ Three different reproducible rRNA gene restriction patterns (ribotypes R-1 to R-3) were obtained among the 195 isolates with the enzyme, HindIII. ^ Colonization factor antigens (CFAs) were identified in 99 (51%) of the 195 ETEC strains studied. ^ Cluster analysis of the observations seen in the four assays all confirmed the five distinct groups of study-year strains of ETEC. Each group had a >95% similarity level of strains within the group and <60% similarity level between the groups. In addition, discriminant analysis of assay variables used in predicting the ETEC strains, reveal a >80% relationship between both the plasmid and rRNA content of ETEC strains and study-year. ^ These findings, based on laboratory observations of the differences in biochemical, antimicrobial susceptibility, plasmid and ribotype content, suggest complex epidemiology for ETEC strains in a population with travelers' diarrhea. The findings of this study may have implications for our understanding of the epidemiology, transmission, treatment, control and prevention of the disease. It has been suggested that an ETEC vaccine for humans should contain the most prevalent CFAs. Therefore, it is important to know the prevalence of these factors in ETEC in various geographical areas. ^ CFAs described in this dissertation may be used in different epidemiological studies in which the prevalence of CFAs and other properties on ETEC will be evaluated. Furthermore, in spite of an intense search in near 200 ETEC isolates for strains that may have clonal relationship, we failed to identify such strains. However, further studies are in progress to construct suitable live vaccine strains and to introduce several of CFAs in the same host organism by recombinant DNA techniques (Dr. Ann-Mari Svennerholm's lab). (Abstract shortened by UMI.)^

The role of conserved region 1 of Escherichia coli sigma-70 in transcription initiation

The sigma (σ) subunit of eubacterial RNA polymerase is essential for initiation of transcription at promoter sites. σ factor directs the RNA polymerase core subunits ( a2bb′ ) to the promoter consensus elements and thereby confers selectivity for transcription initiation. The N-terminal domain (region 1.1) of Escherichia coli σ70 has been shown to inhibit DNA binding by the C-terminal DNA recognition domains when σ is separated from the core subunits. Since DNA recognition by RNA polymerase is the first step in transcription, it seemed plausible that region 1 might also influence initiation processes subsesquent to DNA binding. This study explores the functional roles of regions 1.1 and 1.2 of σ70 in transcription initiation. Analysis in vitro of the transcriptional properties of a series of N-terminally truncated σ70 derivates revealed a critical role for region 1.1 at several key stages of initiation. Deletion of the first 75 to 100 amino acids of σ70 (region 1.1) resulted in both a slow rate of transition from a closed promoter complex to a DNA-strand-separated open complex, as well as a reduced efficiency of transition from the open complex to a transcriptionally active open complex. These effects were partially reversed by addition of a polypeptide containing region 1.1 in trans. Therefore, region 1.1 not only modulates DNA binding but is important for efficient transcription initiation, once a closed complex has formed. A deletion of the first 133 amino acids which removes both regions 1.1 and 1.2 resulted in arrest of initiation at the earliest closed complex, suggesting that region 1.2 is required for open complex formation. Mutagenesis of region 1.1 uncovered a mechanistically important role for isoleucine at position 53 (I53). Substitution of I53 with alanine created a σ factor that associated with the core subunits to form holoenzyme, but the holoenzyme was severely deficient for promoter binding. The I53A phenotype was suppressed in vivo by truncation of five amino acids from the C-terminus of σ 70. These observations are consistent with a model in which σ 70I53A fails to undergo a critical conformational change upon association with the core subunits, which is needed to expose the DNA-binding domains and confer promoter recognition capability upon holoenzyme. To understand the basis of the autoinhibitory properties of the σ70 N-terminal domain, in the absence of core RNA polymerase, a preliminary physical assessment of the interdomain interactions within the σ70 subunit was launched. Results support a model in which N-terminal amino acids are in close proximity to residues in the C-terminus of the σ 70 polypeptide. ^

Protein-protein interaction between phototaxis receptor sensory Rhodopsin I and its transducer HtrI

The molecular complex containing the seven transmembrane helix photoreceptor S&barbelow;ensory R&barbelow;hodopsin I&barbelow; (SRI) and transducer protein HtrI (H&barbelow;alobacterial Transducer for SRI&barbelow;) mediates color-sensitive phototaxis responses in the archaeon Halobacterium salinarum. Orange light causes an attractant response by a one-photon reaction and white light (orange + UV light) a repellent response by a two-photon reaction. Three aspects of SRI-HtrI structure/function and the signal transduction pathway were explored. First, the coupling of HtrI to the photoactive site of SRI was analyzed by mutagenesis and kinetic spectroscopy. Second, SRI-HtrI mutations and suppressors were selected and characterized to elucidate the color-sensing mechanism. Third, the signal relay through the transducer-bound histidine kinase was analyzed using an in vitro reconstitution system with known and newly identified taxis components. ^ Twenty-one mutations on HtrI were introduced by site-directed mutagenesis. Several replacements of charged residues perturbed the photochemical kinetics of SRI which led to the finding of a cluster of residues at the membrane/cytoplasm interface in HtrI electrostatically coupled to the photoactive site of SRI. We found by laser-flash kinetic spectroscopy that the transducer and these residues have specific effects on the light-induced proton transfer between the retinal chromophore and the protein. ^ One of the mutations showed an unusual mutant phenotype we called “inverted” signaling, in which the cell produces a repellent response to normally attractant light. Therefore, this mutant (E56Q of HtrI) had lost the color-discrimination by the SRI-HtrI complex. We used suppressor analysis to better understand the phenotype. Certain suppressors resulted in return of attractant responses to orange light but with inversion of the normally repellent response to white light to an attractant response. To explain this and other results, we formulated the Conformational Shuttling model in which the HtrI-SRI complex is poised in a metastable equilibrium of two conformations shifted in opposite directions by orange and white light. We tested this model by behavioral analysis (computerized cell tracking and motion study) of double mutants of inverting and suppressing mutations and the results confirmed the equilibrium-shift explanation. ^ We developed an in vitro system for measuring the effect of purified transducer on the histidine-kinase CheAH that controls the flagellar motor switch. The rate of kinase autophosphorylation was stimulated >2 fold in the reconstitution of the complete signal transduction system from purified components from H. salinarum. The in vitro assay also showed that the kinase activity was reduced in the absence and in the presence of high levels of linker protein CheWH. (Abstract shortened by UMI.) ^

Role of transducer proteins in photoaxis signaling of Halobacterium salinarum

In Halobacterium salinarum phototaxis is mediated by the visual pigment-like photoreceptors sensory rhodopsin I (SRI) and II (SRII). SRI is a receptor for attractant orange and repellent UV-blue light, and SRII is a receptor for repellent blue-green light, and transmit signals through the membrane-bound transducer proteins HtrI and HtrII, respectively. ^ The primary sequences of HtrI and HtrII predict 2 transmembrane helices (TM1 and TM2) followed by a hydrophilic cytoplasmic domain. HtrII shows an additional large periplasmic domain for chemotactic ligand binding. The cytoplasmic regions are homologous to the adaptation and signaling domains of eubacterial chemotaxis receptors and, like their eubacterial homologs, modulate the transfer of phosphate groups from the histidine protein kinase CheA to the response regulator CheY that in turn controls flagellar motor rotation and the cell's swimming behavior. HtrII and Htrl are dimeric proteins which were predicted to contain carboxylmethylation sites in a 4-helix bundle in their cytoplasmic regions, like eubacterial chemotaxis receptors. ^ The phototaxis transducers of H. salinarum have provided a model for studying receptor/tranducer interaction, adaptation in sensory systems, and the role of membrane molecular complexes in signal transduction. ^ Interaction between the transducer HtrI and the photoreceptor SRI was explored by creating six deletion constructs of HtrI, with progressively shorter cytoplasmic domains. This study confirmed a putative chaperone-like function of HtrI, facilitating membrane insertion or stability of the SRI protein, a phenomenon previously observed in the laboratory, and identified the smallest HtrI fragment containing interaction sites for both the chaperone-like function and SRI photocycle control. The active fragment consisted of the N-terminal 147 residues of the 536-residue HtrI protein, a portion of the molecule predicted to contain the two transmembrane helices and the first ∼20% of the cytoplasmic portion of the protein. ^ Phototaxis and chemotaxis sensory systems adapt to stimuli, thereby signaling only in response to changes in environmental conditions. Observations made in our and in other laboratories and homologies between the halobacterial transducers with the chemoreceptors of enteric bacteria anticipated a role for methylation in adaptation to chemo- and photostimuli. By site directed mutagenesis we identified the methylation sites to be the glutamate pairs E265–E266 in HtrI and E513–E514 in HtrII. Cells containing the unmethylatable transducers are still able to perform phototaxis and adapt to light stimuli. By pulse-chase analysis we found that methanol production from carboxylmethyl group hydrolysis occurs upon specific photo stimulation of unmethylatable HtrI and HtrII and is due to turnover of methyl groups on other transducers. We demonstrated that the turnover in wild-type H. salinarum cells that follows a positive stimulus is CheY-dependent. The CheY-feedback pathway does not require the stimulated transducer to be methylatable and operates globally on other transducers present in the cell. ^ Assembly of signaling molecules into architecturally defined complexes is considered essential in transmission of the signals. The spectroscopic characteristics of SRI were exploited to study the stoichiometric composition in the phototaxis complex SRI-HtrI. A molar ratio of 2.1 HtrI: 1 SRI was obtained, suggesting that only 1 SRI binding site is occupied on the HtrI homodimer. We used gold-immunoelectron microscopy and light fluorescence microscopy to investigate the structural organization and the distribution of other halobacterial transducers. We detected clusters of transducers, usually near the cell's poles, providing a ultrastructural basis for the global effects and intertransducer communication we observe. ^

MONOCLONAL ANTIBODY CHARACTERIZATION OF RAT TRANSFORMATION ASSOCIATED PROTEINS INDUCED BY MOLONEY MURINE SARCOMA VIRUS

By the use of Moloney murine sarcoma virus (Mo-MSV)-induced rat bone tumor (RBT) cells as immunogens, and the hybridoma technique, a mouse hybridoma clone was isolated in Dr. Chan's lab (Chan et al., 1983), which produced a monoclonal antibody, designated MC. MC detected specific antigens in three different Mo-MSV-transformed rat cell lines: 78A1 WRC, RBT and 6M2 (NRK cells infected with the ts110 mutant of Mo-MSV), but not in their untransformed counterparts. These antigens are tentatively termed transformation associated proteins (TAP). In this study, TAP were hypothesized to be the rat specific proteins which are activated by Mo-MSV and play an important role in cellular transformation, and were further investigated. Their properties are summarized as follows: (1) TAP may represent cellular products localized in the cytoplasm of 6M2 cells. (2) The expression of TAP is temperature-sensitive and related to cellular transformation, and probably activated by the v-mos gene products. The optimal temperature for the expression of both P85('gag-mos), the only known viral transforming protein in 6M2 cells, and TAP was 28(DEGREES)C. The expression of both P85('gag-mos) and TAP was proportional to the degree of transformation of 6M2 cells. (3) There were four antigenically-related forms of intracellular TAP (P66, P63, P60 and P58) in 6M2 cells. After synthesis, the 58Kd TAP was probably converted to one of the other three forms. These three polypeptides (P66, P63 and P60) were rapidly converted to two (P68 and P64) and subsequently secreted to the extracellular medium with a 50% secretion rate of 78 min. The conversion of these molecular sizes of TAP is probably related to glycosylation. Inhibition of TAP glycosylation by 0.5 ug/ml of tunicamycin could retard the secretion rate of TAP by 39%. (4) TAP are phosphoproteins, but not associated with any protein kinase activity. (5) TAP have been purified, and found to be mitogenic NRK-2 cells. TAP can bind to the receptors of NRK-2 cells with a K(,d) of 1.4 pM and with about 2 x 10('5) binding sites for TAP per NRK-2 cell. (6) Some weak proteolytic activity was found to associate with purified TAP. ^

HETEROGENEITY IN VIRUS EXPRESSION, TUMORIGENICITY, AND IN VITRO GROWTH PROPERTIES OF DRUG-RESISTANT CLONES OF AN RIII MOUSE MAMMARY TUMOR CELL LINE

Four 8-azaguanine (AG)-resistant and 5-bromodeoxyuridine (BUdR)-resistant clones of a mouse mammary adenocarcinoma cell line, RIII 7387, were developed and analyzed for their tumorigenic properties, in vitro characteristics, and virus expression. These characteristics were analyzed for relationships of any of the cellular parameters and the ability of these lines to produce tumors in syngeneic animals.^ The results of this study demonstrated that the parental line consists of a heterogeneous population of cells. Doubling times, saturation densities, and 2-deoxy-D-glucose uptake varied between sublines. In addition, while all sublines were found to express both B-type and C-type viral antigenic markers, levels of the major B-type and C-type viral proteins varied in the subclones. The sublines also differed markedly in their response to the presence of dexamethasone, glutathione, and insulin in the tissue culture medium.^ Variations in retrovirus expression were convirmed by electron microscopy. Budding and extracellular virus particles were seen in the majority of the cell lines. Virus particles in one of the BUdR-resistant lines, BUD9, were found however, only in inclusions and vacuoles. The AG-resistant subline AGE11 was observed to be rich in intracytoplasmic A particles. The examination of these cell lines for the presence of retroviral RNA-dependent DNA polymerase (RT) activity revealed that some B-type RT activity could be found in the culture fluid of most of the cell lines but that little C-type RT activity could be found suggesting that the C-type virus particles expressed by these RIII clones contain a defective RT.^ Tumor clones also varied in their ability to form tumors in syngeneic RIII mice. Tumor incidence ranged from 50% to 100%. The majority of the tumors regressed within 30 days post infection.^ Statistical analysis indicated that while these clones varied in their characteristics, there was no correlation between the ability of these cell lines to form tumors in syngeneic mice and any of the other characteristics examined.^ These studies have confirmed and extended the growing evidence that tumors, regardless of their natural origin, consist of heterogeneous subpopulations of cells which may vary widely in their in vitro growth behavior, their antigenic expression, and their malignant properties. ^