Amygdala-enriched genes identified by microarray technology are restricted to specific amygdaloid subnuclei

Microarray technology represents a potentially powerful method for identifying cell type- and regionally restricted genes expressed in the brain. Here we have combined a microarray analysis of differential gene expression among five selected brain regions, including the amygdala, cerebellum, hippocampus, olfactory bulb, and periaqueductal gray, with in situ hybridization. On average, 0.3% of the 34,000 genes interrogated were highly enriched in each of the five regions, relative to the others. In situ hybridization performed on a subset of amygdala-enriched genes confirmed in most cases the overall region-specificity predicted by the microarray data and identified additional sites of brain expression not examined on the microarrays. Strikingly, the majority of these genes exhibited boundaries of expression within the amygdala corresponding to cytoarchitectonically defined subnuclei. These results define a unique set of molecular markers for amygdaloid subnuclei and provide tools to genetically dissect their functional roles in different emotional behaviors.

Restricted expression of Kaposi sarcoma-associated herpesvirus (human herpesvirus 8) genes in Kaposi sarcoma.

Kaposi sarcoma (KS) is the leading neoplasm of HIV-infected patients and is also found in several HIV-negative populations. Recently, DNA sequences from a novel herpesvirus, termed KS-associated herpesvirus (KSHV), or human herpesvirus 8 (HHV-8) have been identified within KS tissue from both HIV-positive and HIV-negative cases; infection with this agent has been proposed as a possible factor in the etiology or pathogenesis of the tumor. Here we have examined the pattern of KSHV/HHV-8 gene expression in KS and find it to be highly restricted. We identify and characterize two small transcripts that represent the bulk of the virus-specific RNA transcribed from over 120 kb of the KSHV genome in infected cells. One transcript is predicted to encode a small membrane protein; the other is an unusual polyadenylylated RNA that accumulates in the nucleus to high copy number. This pattern of viral gene expression suggests that most infected cells in KS are latently infected, with lytic viral replication likely restricted to a much smaller subpopulation of cells. These findings have implications for the therapeutic utility of currently available antiviral drugs targeted against the lytic replication cycle.

Analysis of lipopolysaccharide-response genes in B-lineage cells demonstrates that they can have differentiation stage-restricted expression and contain SH2 domains.

Bacterial lipopolysaccharide (LPS) is a potent stimulator of B-cell activation, proliferation, and differentiation. We examined the genetic response of B-lineage cells to LPS via trapping of expressed genes with a gene-trap retrovirus. This analysis showed that expression of only a small fraction of genes is altered during LPS stimulation of B-lineage cells. Isolation of the cellular portion of the trapped LPS-response genes via 5' RACE (rapid amplification of cDNA ends) cloning identified novel genes for all the cloned loci. These novel LPS-response genes were also found to have differentiation stage-restricted expression within the B-lymphoid lineage. That LPS-response genes in B cells also have differentiation stage-restricted expression suggests that these genes may be involved in the control of B-cell function and differentiation, since the known members of this class of genes have frequently been found to play a role in the function and differentiation of B-lineage cells. The isolation of novel members of this class of genes, including a gene that contains a putative SH2 domain, will further increase our understanding of the molecular events involved in the control of B-cell differentiation and function.

Restricted expression of homeobox genes distinguishes fetal from adult human smooth muscle cells.

Smooth muscle cell plasticity is considered a prerequisite for atherosclerosis and restenosis following angioplasty and bypass surgery. Identification of transcription factors that specify one smooth muscle cell phenotype over another therefore may be of major importance in understanding the molecular basis of these vascular disorders. Homeobox genes exemplify one class of transcription factors that could govern smooth muscle cell phenotypic diversity. Accordingly, we screened adult and fetal human smooth muscle cell cDNA libraries with a degenerate oligonucleotide corresponding to a highly conserved region of the homeodomain with the idea that homeobox genes, if present, would display a smooth muscle cell phenotype-dependent pattern of expression. No homeobox genes were detected in the adult human smooth muscle cell library; however, five nonparalogous homeobox genes were uncovered from the fetal library (HoxA5, HoxA11, HoxB1, HoxB7, and HoxC9). Northern blotting of adult and fetal tissues revealed low and restricted expression of all five homeobox genes. No significant differences in transcripts of HoxA5, HoxA11, and HoxB1 were detected between adult or fetal human smooth muscle cells in culture. HoxB7 and HoxC9, however, showed preferential mRNA expression in fetal human smooth muscle cells that appeared to correlate with the age of the donor. This phenotype-dependent expression of homeobox genes was also noted in rat pup versus adult smooth muscle cells. While similar differences in gene expression have been reported between subsets of smooth muscle cells from rat vessels of different-aged animals or clones of rat smooth muscle, our findings represent a demonstration of a transcription factor distinguishing two human smooth muscle cell phenotypes.

Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination

One-third of humans are infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. Sequence analysis of two megabases in 26 structural genes or loci in strains recovered globally discovered a striking reduction of silent nucleotide substitutions compared with other human bacterial pathogens. The lack of neutral mutations in structural genes indicates that M. tuberculosis is evolutionarily young and has recently spread globally. Species diversity is largely caused by rapidly evolving insertion sequences, which means that mobile element movement is a fundamental process generating genomic variation in this pathogen. Three genetic groups of M. tuberculosis were identified based on two polymorphisms that occur at high frequency in the genes encoding catalase-peroxidase and the A subunit of gyrase. Group 1 organisms are evolutionarily old and allied with M. bovis, the cause of bovine tuberculosis. A subset of several distinct insertion sequence IS6110 subtypes of this genetic group have IS6110 integrated at the identical chromosomal insertion site, located between dnaA and dnaN in the region containing the origin of replication. Remarkably, study of ≈6,000 isolates from patients in Houston and the New York City area discovered that 47 of 48 relatively large case clusters were caused by genotypic group 1 and 2 but not group 3 organisms. The observation that the newly emergent group 3 organisms are associated with sporadic rather than clustered cases suggests that the pathogen is evolving toward a state of reduced transmissability or virulence.

Mouse Eya genes are expressed during limb tendon development and encode a transcriptional activation function

Vertebrate limb tendons are derived from connective cells of the lateral plate mesoderm. Some of the developmental steps leading to the formation of vertebrate limb tendons have been previously identified; however, the molecular mechanisms responsible for tendinous patterning and maintenance during embryogenesis are largely unknown. The eyes absent (eya) gene of Drosophila encodes a novel nuclear protein of unknown molecular function. Here we show that Eya1 and Eya2, two mouse homologues of Drosophila eya, are expressed initially during limb development in connective tissue precursor cells. Later in limb development, Eya1 and Eya2 expression is associated with cell condensations that form different sets of limb tendons. Eya1 expression is largely restricted to flexor tendons, while Eya2 is expressed in the extensor tendons and ligaments of the phalangeal elements of the limb. These data suggest that Eya genes participate in the patterning of the distal tendons of the limb. To investigate the molecular functions of the Eya gene products, we have analyzed whether the highly divergent PST (proline-serine-threonine)-rich N-terminal regions of Eya1–3 function as transactivation domains. Our results demonstrate that Eya gene products can act as transcriptional activators, and they support a role for this molecular function in connective tissue patterning.

The diversity and evolutionary relationships of the pregnancy-associated glycoproteins, an aspartic proteinase subfamily consisting of many trophoblast-expressed genes

The pregnancy-associated glycoproteins (PAGs) are structurally related to the pepsins, thought to be restricted to the hooved (ungulate) mammals and characterized by being expressed specifically in the outer epithelial cell layer (chorion/trophectoderm) of the placenta. At least some PAGs are catalytically inactive as proteinases, although each appears to possess a cleft capable of binding peptides. By cloning expressed genes from ovine and bovine placental cDNA libraries, by Southern genomic blotting, by screening genomic libraries, and by using PCR to amplify portions of PAG genes from genomic DNA, we estimate that cattle, sheep, and most probably all ruminant Artiodactyla possess many, possibly 100 or more, PAG genes, many of which are placentally expressed. The PAGs are highly diverse in sequence, with regions of hypervariability confined largely to surface-exposed loops. Nonsynonymous (replacement) mutations in the regions of the genes coding for these hypervariable loop segments have accumulated at a higher rate than synonymous (silent) mutations. Construction of distance phylograms, based on comparisons of PAG and related aspartic proteinase amino acid sequences, suggests that much diversification of the PAG genes occurred after the divergence of the Artiodactyla and Perissodactyla, but that at least one gene is represented outside the hooved species. The results also suggest that positive selection of duplicated genes has acted to provide considerable functional diversity among the PAGs, whose presence at the interface between the placenta and endometrium and in the maternal circulation indicates involvement in fetal–maternal interactions.

Duplicated genes evolve independently after polyploid formation in cotton

Of the many processes that generate gene duplications, polyploidy is unique in that entire genomes are duplicated. This process has been important in the evolution of many eukaryotic groups, and it occurs with high frequency in plants. Recent evidence suggests that polyploidization may be accompanied by rapid genomic changes, but the evolutionary fate of discrete loci recently doubled by polyploidy (homoeologues) has not been studied. Here we use locus-specific isolation techniques with comparative mapping to characterize the evolution of homoeologous loci in allopolyploid cotton (Gossypium hirsutum) and in species representing its diploid progenitors. We isolated and sequenced 16 loci from both genomes of the allopolyploid, from both progenitor diploid genomes and appropriate outgroups. Phylogenetic analysis of the resulting 73.5 kb of sequence data demonstrated that for all 16 loci (14.7 kb/genome), the topology expected from organismal history was recovered. In contrast to observations involving repetitive DNAs in cotton, there was no evidence of interaction among duplicated genes in the allopolyploid. Polyploidy was not accompanied by an obvious increase in mutations indicative of pseudogene formation. Additionally, differences in rates of divergence among homoeologues in polyploids and orthologues in diploids were indistinguishable across loci, with significant rate deviation restricted to two putative pseudogenes. Our results indicate that most duplicated genes in allopolyploid cotton evolve independently of each other and at the same rate as those of their diploid progenitors. These indications of genic stasis accompanying polyploidization provide a sharp contrast to recent examples of rapid genomic evolution in allopolyploids.

Inhibition of MHC class I-restricted antigen presentation by γ2-herpesviruses

The γ-herpesviruses, in contrast to the α- and β-herpesviruses, are not known to inhibit antigen presentation to CD8+ cytotoxic T lymphocytes (CTLs) during lytic cycle replication. However, murine γ-herpesvirus 68 causes a chronic lytic infection in CD4+ T cell-deficient mice despite the persistence of a substantial CTL response, suggesting that CTL evasion occurs. Here we show that, distinct from host protein synthesis shutoff, γ-herpesvirus 68 down-regulates surface MHC class I expression on lytically infected fibroblasts and inhibits their recognition by antigen-specific CTLs. The viral K3 gene, encoding a zinc-finger-containing protein, dramatically reduced the half-life of nascent class I molecules and the level of surface MHC class I expression and was by itself sufficient to block antigen presentation. The homologous K3 and K5 genes of the related Kaposi's sarcoma-associated virus also inhibited antigen presentation and decreased cell surface expression of HLA class I antigens. Thus it appears that an immune evasion strategy shared by at least two γ-herpesviruses allows continued lytic infection in the face of strong CTL immunity.

Discovery of genes involved with learning and memory: An experimental synthesis of Hirschian and Benzerian perspectives

The biological bases of learning and memory are being revealed today with a wide array of molecular approaches, most of which entail the analysis of dysfunction produced by gene disruptions. This perspective derives both from early “genetic dissections” of learning in mutant Drosophila by Seymour Benzer and colleagues and from earlier behavior-genetic analyses of learning and in Diptera by Jerry Hirsch and coworkers. Three quantitative-genetic insights derived from these latter studies serve as guiding principles for the former. First, interacting polygenes underlie complex traits. Consequently, learning/memory defects associated with single-gene mutants can be quantified accurately only in equilibrated, heterogeneous genetic backgrounds. Second, complex behavioral responses will be composed of genetically distinct functional components. Thus, genetic dissection of complex traits into specific biobehavioral properties is likely. Finally, disruptions of genes involved with learning/memory are likely to have pleiotropic effects. As a result, task-relevant sensorimotor responses required for normal learning must be assessed carefully to interpret performance in learning/memory experiments. In addition, more specific conclusions will be obtained from reverse-genetic experiments, in which gene disruptions are restricted in time and/or space.

Light-Regulated Transcription of Genes Encoding Peridinin Chlorophyll a Proteins and the Major Intrinsic Light-Harvesting Complex Proteins in the Dinoflagellate Amphidinium carterae Hulburt (Dinophycae)1 : Changes in Cytosine Methylation Accompany Photoadaptation

In the dinoflagellate Amphidinium carterae, photoadaptation involves changes in the transcription of genes encoding both of the major classes of light-harvesting proteins, the peridinin chlorophyll a proteins (PCPs) and the major a/c-containing intrinsic light-harvesting proteins (LHCs). PCP and LHC transcript levels were increased up to 86- and 6-fold higher, respectively, under low-light conditions relative to cells grown at high illumination. These increases in transcript abundance were accompanied by decreases in the extent of methylation of CpG and CpNpG motifs within or near PCP- and LHC-coding regions. Cytosine methylation levels in A. carterae are therefore nonstatic and may vary with environmental conditions in a manner suggestive of involvement in the regulation of gene expression. However, chemically induced undermethylation was insufficient in activating transcription, because treatment with two methylation inhibitors had no effect on PCP mRNA or protein levels. Regulation of gene activity through changes in DNA methylation has traditionally been assumed to be restricted to higher eukaryotes (deuterostomes and green plants); however, the atypically large genomes of dinoflagellates may have generated the requirement for systems of this type in a relatively “primitive” organism. Dinoflagellates may therefore provide a unique perspective on the evolution of eukaryotic DNA-methylation systems.

The function of herpes simplex virus genes: a primer for genetic engineering of novel vectors.

Herpes simplex virus vectors are being developed for delivery and expression of human genes to the central nervous system, selective destruction of cancer cells, and as carriers for genes encoding antigens that induce protective immunity against infectious agents. Vectors constructed to meet these objectives must differ from wild-type virus with respect to host range, reactivation from latency, and expression of viral genes. The vectors currently being developed are (i) helper free amplicons, (ii) replication defective viruses, and (iii) genetically engineered replication competent viruses with restricted host range. Whereas the former two types of vectors require stable, continuous cell lines expressing viral genes for their replication, the replication competent viruses will replicate on approved primary human cell strains.

Host range restrictions of oncogenes: myc genes transform avian but not mammalian cells and mht/raf genes transform mammalian but not avian cells.

The host range of retroviral oncogenes is naturally limited by the host range of the retroviral vector. The question of whether the transforming host range of retroviral oncogenes is also restricted by the host species has not been directly addressed. Here we have tested in avian and murine host species the transforming host range of two retroviral onc genes, myc of avian carcinoma viruses MH2 and MC29 and mht/raf of avian carcinoma virus MH2 and murine sarcoma virus MSV 3611. Virus vector-mediated host restriction was bypassed by recombining viral oncogenes with retroviral vectors that can readily infect the host to be tested. It was found that, despite high expression, transforming function of retroviral myc genes is restricted to avian cells, and that of retroviral mht/raf genes is restricted to murine cells. Since retroviral oncogenes encode the same proteins as certain cellular genes, termed protooncogenes, our data must also be relevant to the oncogene hypothesis of cancer. According to this hypothesis, cancer is caused by mutation of protooncogenes. Because protooncogenes are conserved in evolution and are presumed to have conserved functions, the oncogene hypothesis assumes no host range restriction of transforming function. For example, mutated human proto-myc is postulated to cause Burkitt lymphoma, because avian retroviruses with myc genes cause cancer in birds. But there is no evidence that known mutated protooncogenes can transform human cells. The findings reported here indicate that host range restriction appears to be one of the reasons (in addition to insufficient transcriptional activation) why known, mutated protooncogenes lack transforming function in human cells.

An induction gene trap screen in embryonic stem cells: Identification of genes that respond to retinoic acid in vitro.

We have developed a novel induction gene trap approach that preselects in vitro for integrations into genes that lie downstream of receptor/ligand-mediated signaling pathways. Using this approach, we have identified 20 gene trap integrations in embryonic stem cells, 9 of which were induced and 11 of which were repressed after exposure to exogenous retinoic acid (RA). All but one of these integrations showed unique spatially restricted or tissue-specific patterns of expression between 8.5 and 11.5 days of embryogenesis. Interestingly, expression was observed in tissues that are affected by alterations in RA levels during embryogenesis. Sequence analysis of fusion transcripts from six integrations revealed five novel gene sequences and the previously identified protooncogene c-fyn. To date, germ-line transmission and breeding has uncovered one homozygous embryonic lethal and three homozygous viable insertions. These studies demonstrate the potential of this induction gene trap approach for identifying and mutating genes downstream of signal transduction pathways.

Homeobox-containing gene transiently expressed in a spatially restricted pattern in the early sea urchin embryo.

In the sea urchin embryo, the lineage founder cells whose polyclonal progenies will give rise to five different territories are segregated at the sixth division. To investigate the mechanisms by which the fates of embryonic cells are first established, we looked for temporal and spatial expression of homeobox genes in the very early cleavage embryos. We report evidence that PlHbox12, a paired homeobox-containing gene, is expressed in the embryo from the 4-cell stage. The abundance of the transcripts reaches its maximum when the embryo has been divided into the five polyclonal territories--namely at the 64-cell stage--and it abruptly declines at later stages of development. Blastomere dissociation experiments indicate that maximal expression of PlHbox12 is dependent on intercellular interactions, thus suggesting that signal transduction mechanisms are responsible for its transcriptional activation in the early cleavage embryo. Spatial expression of PlHbox12 was determined by whole-mount in situ hybridization. PlHbox12 transcripts in embryos at the fourth, fifth, and sixth divisions seem to be restricted to the conditionally specified ectodermal lineages. These results suggest a possible role of the PlHbox12 gene in the early events of cell specification of the presumptive ectodermal territories.