Genetic basis of tolerance to O2 deprivation in Drosophila melanogaster

The ability to tolerate a low-O2 environment varies widely among species in the animal kingdom. Some animals, such as Drosophila melanogaster, can tolerate anoxia for prolonged periods without apparent tissue injury. To determine the genetic basis of the cellular responses to low O2, we performed a genetic screen in Drosophila to identify loci that are responsible for anoxia resistance. Four X-linked, anoxia-sensitive mutants belonging to three complementation groups were isolated after screening more than 10,000 mutagenized flies. The identified recessive and dominant mutations showed marked delay in recovery from O2 deprivation. In addition, electrophysiologic studies demonstrated that polysynaptic transmission in the central nervous system of the mutant flies was abnormally long during recovery from anoxia. These studies show that anoxic tolerance can be genetically dissected.

Identification of a cellular receptor for subgroup E avian leukosis virus

Genetic studies in chickens and receptor interference experiments have indicated that avian leukosis virus (ALV)-E may utilize a cellular receptor related to the receptor for ALV-B and ALV-D. Recently, we cloned CAR1, a tumor necrosis factor receptor (TNFR)-related protein, that serves as a cellular receptor for ALV-B and ALV-D. To determine whether the cellular receptor for ALV-E is a CAR1-like protein, a cDNA library was made from turkey embryo fibroblasts (TEFs), which are susceptible to ALV-E infection, but not to infection by ALV-B and ALV-D. The cDNA library was screened with a radioactively labeled CAR1 cDNA probe, and clones that hybridized with the probe were isolated. A 2.3-kb cDNA clone was identified that conferred susceptibility to ALV-E infection, but not to ALV-B infection, when expressed in transfected human 293 cells. The functional cDNA clone is predicted to encode a 368 amino acid protein with significant amino acid similarity to CAR1. Like CAR1, the TEF protein is predicted to have two extracellular TNFR-like cysteine-rich domains and a putative death domain similar to those of TNFR I and Fas. Flow cytometric analysis and immunoprecipitation experiments demonstrated specific binding between the TEF CAR1-related protein and an immunoadhesin composed of the surface (SU) envelope protein of subgroup E (RAV-0) virus fused to the constant region of a rabbit immunoglobulin. These two activities of the TEF CAR1-related protein, specific binding to ALV-E SU and permitting entry only of ALV-E, have unambiguously identified this protein as a cellular receptor specific for subgroup E ALV.

Efficient loading of HLA-DR with a T helper epitope by genetic exchange of CLIP

The HLA class II-associated invariant chain (Ii)-derived peptide (CLIP) occupies the peptide binding groove during assembly in the endoplasmic reticulum, travels with HLA class II to endosomal compartments, and is subsequently released to allow binding of antigenic peptides. We investigated whether the exchange of CLIP with a known T helper epitope at the DNA level would lead to efficient loading of this helper epitope onto HLA class II. For this purpose, a versatile Ii-encoding expression vector was created in which CLIP can be replaced with a helper epitope of choice. Upon supertransfection of HLA-DR1-transfected 293 cells with an Ii vector encoding a known T helper epitope (HA307–319), predominantly length variants of this epitope were detected in association with the HLA-DR1 molecules of these cells. Moreover, this transfectant was efficiently recognized by a peptide-specific T helper clone (HA1.7). The results suggest that this type of Ii vector can be used to create potent class II+ cellular vaccines in which defined T cell epitopes are continuously synthesized.

Effect of cellular filamentation on adventurous and social gliding motility of Myxococcus xanthus

Filamentous bacterial cells often provide biological information that is not readily evident in normal-size cells. In this study, the effect of cellular filamentation on gliding motility of Myxococcus xanthus, a Gram-negative social bacterium, was investigated. Elongation of the cell body had different effects on adventurous and social motility of M. xanthus. The rate of A-motility was insensitive to cell-body elongation whereas the rate of S-motility was reduced dramatically as the cell body got longer, indicating that these two motility systems work in different ways. The study also showed that filamentous wild-type cells glide smoothly with relatively straight, long cell bodies. However, filamentous cells of certain social motility mutants showed zigzag, tangled cell bodies on a solid surface, apparently a result of a lack of coordination between different fragments within the filaments. Further genetic and biochemical analyses indicated that the uncoordinated movements of these mutant filaments were correlated with the absence of cell surface fibril materials, indicating a possible new function for fibrils.

Genetic instability of cancer cells is proportional to their degree of aneuploidy

Genetic and phenotypic instability are hallmarks of cancer cells, but their cause is not clear. The leading hypothesis suggests that a poorly defined gene mutation generates genetic instability and that some of many subsequent mutations then cause cancer. Here we investigate the hypothesis that genetic instability of cancer cells is caused by aneuploidy, an abnormal balance of chromosomes. Because symmetrical segregation of chromosomes depends on exactly two copies of mitosis genes, aneuploidy involving chromosomes with mitosis genes will destabilize the karyotype. The hypothesis predicts that the degree of genetic instability should be proportional to the degree of aneuploidy. Thus it should be difficult, if not impossible, to maintain the particular karyotype of a highly aneuploid cancer cell on clonal propagation. This prediction was confirmed with clonal cultures of chemically transformed, aneuploid Chinese hamster embryo cells. It was found that the higher the ploidy factor of a clone, the more unstable was its karyotype. The ploidy factor is the quotient of the modal chromosome number divided by the normal number of the species. Transformed Chinese hamster embryo cells with a ploidy factor of 1.7 were estimated to change their karyotype at a rate of about 3% per generation, compared with 1.8% for cells with a ploidy factor of 0.95. Because the background noise of karyotyping is relatively high, the cells with low ploidy factor may be more stable than our method suggests. The karyotype instability of human colon cancer cell lines, recently analyzed by Lengnauer et al. [Lengnauer, C., Kinzler, K. W. & Vogelstein, B. (1997) Nature (London) 386, 623–627], also corresponds exactly to their degree of aneuploidy. We conclude that aneuploidy is sufficient to explain genetic instability and the resulting karyotypic and phenotypic heterogeneity of cancer cells, independent of gene mutation. Because aneuploidy has also been proposed to cause cancer, our hypothesis offers a common, unique mechanism of altering and simultaneously destabilizing normal cellular phenotypes.

Developmental signal transduction pathways uncovered by genetic suppressors

We have found conditions for saturation mutagenesis by restriction enzyme mediated integration that result in plasmid tagging of disrupted genes. Using this method we selected for mutations in genes that act at checkpoints downstream of the intercellular signaling system that controls encapsulation in Dictyostelium discoideum. One of these genes, mkcA, is a member of the mitogen-activating protein kinase cascade family while the other, regA, is a novel bipartite gene homologous to response regulators in one part and to cyclic nucleotide phosphodiesterases in the other part. Disruption of either of these genes results in partial suppression of the block to spore formation resulting from the loss of the prestalk genes, tagB and tagC. The products of the tag genes have conserved domains of serine proteases attached to ATP-driven transporters, suggesting that they process and export peptide signals. Together, these genes outline an intercellular communication system that coordinates organismal shape with cellular differentiation during development.

Manipulating the genetic identity and biochemical surface properties of individual cells with electric-field-induced fusion

A method for cell–cell and cell–liposome fusion at the single-cell level is described. Individual cells or liposomes were first selected and manipulated either by optical trapping or by adhesion to a micromanipulator-controlled ultramicroelectrode. Spatially selective fusion of the cell–cell or cell–liposome pair was achieved by the application of a highly focused electric field through a pair of 5-μm o.d. carbon-fiber ultramicroelectrodes. The ability to fuse together single cells opens new possibilities in the manipulation of the genetic and cellular makeup of individual cells in a controlled manner. In the study of cellular networks, for example, the alteration of the biochemical identity of a selected cell can have a profound effect on the behavior of the entire network. Fusion of a single liposome with a target cell allows the introduction of the liposomal content into the cell interior as well as the addition of lipids and membrane proteins onto the cell surface. This cell–liposome fusion represents an approach to the manipulation of the cytoplasmic contents and surface properties of single cells. As an example, we have introduced a membrane protein (γ-glutamyltransferase) reconstituted in liposomes into the cell plasma membrane.

Quantitative assessment of enzyme specificity in vivo: P2 recognition by Kex2 protease defined in a genetic system

The specificity of the yeast proprotein-processing Kex2 protease was examined in vivo by using a sensitive, quantitative assay. A truncated prepro-α-factor gene encoding an α-factor precursor with a single α-factor repeat was constructed with restriction sites for cassette mutagenesis flanking the single Kex2 cleavage site (-SLDKR↓EAEA-). All of the 19 substitutions for the Lys (P2) residue in the cleavage site were made. The wild-type and mutant precursors were expressed in a yeast strain lacking the chromosomal genes encoding Kex2 and prepro-α-factor. Cleavage of the 20 sites by Kex2, expressed at the wild-type level, was assessed by using a quantitative-mating assay with an effective range greater than six orders of magnitude. All substitutions for Lys at P2 decreased mating, from 2-fold for Arg to >106-fold for Trp. Eviction of the Kex2-encoding plasmid indicated that cleavage of mutant sites by other cellular proteases was not a complicating factor. Mating efficiencies of strains expressing the mutant precursors correlated well with the specificity (kcat/KM) of purified Kex2 for comparable model peptide substrates, validating the in vivo approach as a quantitative method. The results support the conclusion that KM, which is heavily influenced by the nature of the P2 residue, is a major determinant of cleavage efficiency in vivo. P2 preference followed the rank order: Lys > Arg > Thr > Pro > Glu > Ile > Ser > Ala > Asn > Val > Cys > AsP > Gln > Gly > His > Met > Leu > Tyr > Phe > Trp.

A yeast genetic system for selecting small molecule inhibitors of protein–protein interactions in nanodroplets

Cellular processes are mediated by complex networks of molecular interactions. Dissection of their role most commonly is achieved by using genetic mutations that alter, for example, protein–protein interactions. Small molecules that accomplish the same result would provide a powerful complement to the genetic approach, but it generally is believed that such molecules are rare. There are several natural products, however, that illustrate the feasibility of this approach. Split-pool synthesis now provides a simple mechanical means to prepare vast numbers of complex, even natural product-like, molecules individually attached to cell-sized polymer beads. Here, we describe a genetic system compatible with split-pool synthesis that allows the detection of cell-permeable, small molecule inhibitors of protein–protein interactions in 100- to 200-nl cell culture droplets, prepared by a recently described technique that arrays large numbers of such droplets. These “nanodroplets” contain defined media, cells, and one or more beads containing ≈100 pmol of a photoreleasable small molecule and a controlled number of cells. The engineered Saccharomyces cerevisiae cells used in this study express two interacting proteins after induction with galactose whose interaction results in cell death in the presence of 5-fluoroorotic acid (inducible reverse two-hybrid assay). Disruption of the interaction by a small molecule allows growth, and the small molecule can be introduced into the system hours before induction of the toxic interaction. We demonstrate that the interaction between the activin receptor R1 and the immunophilin protein FKBP12 can be disrupted by the small molecule FK506 at nanomolar concentrations in nanodroplets. This system should provide a general method for selecting cell-permeable ligands that can be used to study the relevance of protein–protein interactions in living cells or organisms.

The role of the D2 dopamine receptor (D2R) in A2A adenosine receptor (A2AR)-mediated behavioral and cellular responses as revealed by A2A and D2 receptor knockout mice

The A2AR is largely coexpressed with D2Rs and enkephalin mRNA in the striatum where it modulates dopaminergic activity. Activation of the A2AR antagonizes D2R-mediated behavioral and neurochemical effects in the basal ganglia through a mechanism that may involve direct A2AR–D2R interaction. However, whether the D2R is required for the A2AR to exert its neural function is an open question. In this study, we examined the role of D2Rs in A2AR-induced behavioral and cellular responses, by using genetic knockout (KO) models (mice deficient in A2ARs or D2Rs or both). Behavioral analysis shows that the A2AR agonist 2–4-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine reduced spontaneous as well as amphetamine-induced locomotion in both D2 KO and wild-type mice. Conversely, the nonselective adenosine antagonist caffeine and the A2AR antagonist 8-(3-chlorostyryl)caffeine produced motor stimulation in mice lacking the D2R, although the stimulation was significantly attentuated. At the cellular level, A2AR inactivation counteracted the increase in enkephalin expression in striatopallidal neurons caused by D2R deficiency. Consistent with the D2 KO phenotype, A2AR inactivation partially reversed both acute D2R antagonist (haloperidol)-induced catalepsy and chronic haloperidol-induced enkephalin mRNA expression. Together, these results demonstrate that A2ARs elicit behavioral and cellular responses despite either the genetic deficiency or pharmacological blockade of D2Rs. Thus, A2AR-mediated neural functions are partially independent of D2Rs. Moreover, endogenous adenosine acting at striatal A2ARs may be most accurately viewed as a facilitative modulator of striatal neuronal activity rather than simply as an inhibitory modulator of D2R neurotransmission.

Ribonucleoprotein infrastructure regulating the flow of genetic information between the genome and the proteome

Following transcription and splicing, each mRNA of a mammalian cell passes into the cytoplasm where its fate is in the hands of a complex network of ribonucleoproteins (mRNPs). The success or failure of a gene to be expressed depends on the performance of this mRNP infrastructure. The entry, gating, processing, and transit of each mRNA through an mRNP network helps determine the composition of a cell's proteome. The machinery that regulates storage, turnover, and translational activation of mRNAs is not well understood, in part, because of the heterogeneous nature of mRNPs. Recently, subsets of cellular mRNAs clustered as members of mRNP complexes have been identified by using antibodies reactive with RNA-binding proteins, including ELAV/Hu, eIF-4E, and poly(A)-binding proteins. Cytoplasmic ELAV/Hu proteins are involved in the stability and translation of early response gene (ERG) transcripts and are expressed predominately in neurons. mRNAs recovered from ELAV/Hu mRNP complexes were found to have similar sequence elements, suggesting a common structural linkage among them. This approach opens the possibility of identifying transcripts physically clustered in vivo that may have similar fates or functions. Moreover, the proteins encoded by physically organized mRNAs may participate in the same biological process or structural outcome, not unlike operons and their polycistronic mRNAs do in prokaryotic organisms. Our goal is to understand the organization and flow of genetic information on an integrative systems level by analyzing the collective properties of proteins and mRNAs associated with mRNPs in vivo.

Id1 regulation of cellular senescence through transcriptional repression of p16/Ink4a

The Id family of helix–loop–helix (HLH) transcriptional regulatory proteins does not possess a basic DNA-binding domain and functions as a negative regulator of basic HLH transcription factors. Id proteins coordinate cell growth and differentiation pathways within mammalian cells and have been shown to regulate G1-S cell-cycle transitions. Although much recent data has implicated Id1 in playing a critical role in modulating cellular senescence, no direct genetic evidence has been reported to substantiate such work. Here we show that Id1-null primary mouse embryo fibroblasts undergo premature senescence despite normal growth profiles at early passage. These cells possess increased expression of the tumor-suppressor protein p16/Ink4a but not p19/ARF, and have decreased cyclin-dependent kinase (cdk) 2 and cdk4 kinase activity. We also show that Id1 is able to directly inhibit p16/Ink4a but not p19/ARF promoter activity via its HLH domain, and that Id1inhibits transcriptional activation at E-boxes within the p16/Ink4a promoter. Our data provide, to our knowledge, the first genetic evidence for a role for Id1 as an inhibitor of cellular senescence and suggest that Id1 functions to delay cellular senescence through repression of p16/Ink4a. Because epigenetic and genetic abrogation of p16/Ink4a function has been implicated in the evolution of several human malignancies, we propose that transcriptional regulation of p16/Ink4a may also provide a mechanism for the dysregulation of normal cellular growth controls during the evolution of human malignancies.

Use of genetic suppressor elements to dissect distinct biological effects of separate p53 domains.

p53 is a multifunctional tumor suppressor protein involved in the negative control of cell growth. Mutations in p53 cause alterations in cellular phenotype, including immortalization, neoplastic transformation, and resistance to DNA-damaging drugs. To help dissect distinct functions of p53, a set of genetic suppressor elements (GSEs) capable of inducing different p53-related phenotypes in rodent embryo fibroblasts was isolated from a retroviral library of random rat p53 cDNA fragments. All the GSEs were 100-300 nucleotides long and were in the sense orientation. They fell into four classes, corresponding to the transactivator (class I), DNA-binding (class II), and C-terminal (class III) domains of the protein and the 3'-untranslated region of the mRNA (class IV). GSEs in all four classes promoted immortalization of primary cells, but only members of classes I and III cooperated with activated ras to transform cells, and only members of class III conferred resistance to etoposide and strongly inhibited transcriptional transactivation by p53. These observations suggest that processes related to control of senescence, response to DNA damage, and transformation involve different functions of the p53 protein and furthermore indicate a regulatory role for the 3'-untranslated region of p53 mRNA.

Irreversibility of cellular aging and neoplastic transformation: a clonal analysis.

Prolonged incubation of NIH 3T3 cells under the growth constraint of confluence results in a persistent impairment of proliferation when the cells are subcultured at low density and a greatly increased probability of neoplastic transformation in assays for transformation. These properties, along with the large accumulation of age pigment bodies in the confluent cells, are cardinal cellular characteristics of aging in organisms and validate the system as a model of cellular aging. Two cultures labeled alpha and beta were obtained after prolonged confluence; both were dominated by cells that were both slowed in growth at low population density and enhanced in growth capacity at high density, a marker of neoplastic transformation. An experiment was designed to study the reversibility of these age-related properties by serial subculture at low density of the two uncloned cultures and their progeny clones derived from assuredly single cells. Both uncloned cultures had many transformed cells and a reduced growth rate on subculture. Serial subculture resulted in a gradual increase in growth rates of both populations, but a reversal of transformation only in the alpha population. The clones originating from both populations varied in the degree of growth impairment and neoplastic transformation. None of the alpha clones increased in growth rate on low density passage nor did the transformed clones among them revert to normal growth behavior. The fastest growing beta clone was originally slower than the control clone, but caught up to it after four weekly subcultures. The other beta clones retained their reduced growth rates. Four of the five beta clones, including the fastest grower, were transformed, and none reverted on subculture. We conclude that the apparent reversal of impaired growth and transformation in the uncloned parental alpha population resulted from the selective growth at low density of fast growing nontransformed clones. The reversal of impaired growth in the uncloned parental beta population was also the result of selective growth of fast growing clones, but in this case they were highly transformed so no apparent reversal of transformation occurred. The clonal results indicate that neither the impaired growth nor the neoplastic transformation found in aging cells is reversible. We discuss the possible contribution of epigenetic and genetic processes to these irreversible changes.

Cellular aging, destabilization, and cancer.

Three major characteristics of aging in animals are a slowdown of cell proliferation, an increase in residual bodies associated with age pigments, and a marked increase in the likelihood of neoplastic transformation. The 28 L subline of the NIH 3T3 line of mouse embryo fibroblasts exhibits all these characteristics when held at confluence for extended periods. The impairment of proliferation is the first behavioral characteristic detected in low density subcultures from the confluent cultures, and it persists through many cell generations of exponential multiplication. There is an equal degree of growth impairment among replicate cultures (lineages) recovered after each of 2 successive rounds of confluence, although heterogeneity appears after the third round. The growth impairment pervades the entire cell population of each lineage. The degree and duration of impairment increase with repeated rounds of confluence. A marked increase of residual bodies characteristic of age pigments occurs in the cytoplasm of all the cells kept under prolonged confluence. Neoplastic transformation first appears as foci of multilayered cells on a monolayered background of nontransformed cells. The transformed cells arise at different times in the lineages and originate from a very small fraction of the population. The transformed cells selectively overgrow the entire population in successive rounds of confluence leading to an increase in saturation density of each lineage at different times. Under cloning conditions, isolated colonies of transformed cells develop more slowly than colonies of nontransformed cells but eventually reach a higher population density. The regularity of persistent growth impairment among the lineages and the appearance of large numbers of residual bodies in all the cells of each population are more characteristic of an epigenetic process than of specific local mutations. although random chromosomal lesions cannot be ruled out. By contrast, the low frequency and stochastic character of neoplastic transformation are consistent with a conventional genetic origin. The advent in long-term confluent NIH 3T3 cultures of three cardinal characteristics of cellular aging in vivo recommends it as a model for aging cells.