Recurrent invasion and extinction of a selfish gene

Homing endonuclease genes show super-Mendelian inheritance, which allows them to spread in populations even when they are of no benefit to the host organism. To test the idea that regular horizontal transmission is necessary for the long-term persistence of these genes, we surveyed 20 species of yeasts for the ω-homing endonuclease gene and associated group I intron. The status of ω could be categorized into three states (functional, nonfunctional, or absent), and status was not clustered on the host phylogeny. Moreover, the phylogeny of ω differed significantly from that of the host, strong evidence of horizontal transmission. Further analyses indicate that horizontal transmission is more common than transposition, and that it occurs preferentially between closely related species. Parsimony analysis and coalescent theory suggest that there have been 15 horizontal transmission events in the ancestry of our yeast species, through simulations indicate that this value is probably an underestimate. Overall, the data support a cyclical model of invasion, degeneration, and loss, followed by reinvasion, and each of these transitions is estimated to occur about once every 2 million years. The data are thus consistent with the idea that frequent horizontal transmission is necessary for the long-term persistence of homing endonuclease genes, and further, that this requirement limits these genes to organisms with easily accessible germ lines. The data also show that mitochondrial DNA sequences are transferred intact between yeast species; if other genes do not show such high levels of horizontal transmission, it would be due to lack of selection, rather than lack of opportunity.

Restriction-modification gene complexes as selfish gene entities: Roles of a regulatory system in their establishment, maintenance, and apoptotic mutual exclusion

We have reported some type II restriction-modification (RM) gene complexes on plasmids resist displacement by an incompatible plasmid through postsegregational host killing. Such selfish behavior may have contributed to the spread and maintenance of RM systems. Here we analyze the role of regulatory genes (C), often found linked to RM gene complexes, in their interaction with the host and the other RM gene complexes. We identified the C gene of EcoRV as a positive regulator of restriction. A C mutation eliminated postsegregational killing by EcoRV. The C system has been proposed to allow establishment of RM systems in new hosts by delaying the appearance of restriction activity. Consistent with this proposal, bacteria preexpressing ecoRVC were transformed at a reduced efficiency by plasmids carrying the EcoRV RM gene complex. Cells carrying the BamHI RM gene complex were transformed at a reduced efficiency by a plasmid carrying a PvuII RM gene complex, which shares the same C specificity. The reduction most likely was caused by chromosome cleavage at unmodified PvuII sites by prematurely expressed PvuII restriction enzyme. Therefore, association of the C genes of the same specificity with RM gene complexes of different sequence specificities can confer on a resident RM gene complex the capacity to abort establishment of a second, incoming RM gene complex. This phenomenon, termed “apoptotic mutual exclusion,” is reminiscent of suicidal defense against virus infection programmed by other selfish elements. pvuIIC and bamHIC genes define one incompatibility group of exclusion whereas ecoRVC gene defines another.

The FEZ1 gene at chromosome 8p22 encodes a leucine-zipper protein, and its expression is altered in multiple human tumors

Alterations of human chromosome 8p occur frequently in many tumors. We identified a 1.5-Mb common region of allelic loss on 8p22 by allelotype analysis. cDNA selection allowed isolation of several genes, including FEZ1. The predicted Fez1 protein contained a leucine-zipper region with similarity to the DNA-binding domain of the cAMP-responsive activating-transcription factor 5. RNA blot analysis revealed that FEZ1 gene expression was undetectable in more than 60% of epithelial tumors. Mutations were found in primary esophageal cancers and in a prostate cancer cell line. Transcript analysis from several FEZ1-expressing tumors revealed truncated mRNAs, including a frameshift. Alteration and inactivation of the FEZ1 gene may play a role in various human tumors.

Loss of transformed phenotype in cancer cells by overexpression of the uteroglobin gene

Uteroglobin (UG) is a multifunctional, secreted protein that has receptor-mediated functions. The human UG (hUG) gene is mapped to chromosome 11q12.2–13.1, a region frequently rearranged or deleted in many cancers. Although high levels of hUG expression are characteristic of the mucosal epithelia of many organs, hUG expression is either drastically reduced or totally absent in adenocarcinomas and in viral-transformed epithelial cells derived from the same organs. In agreement with these findings, in an ongoing study to evaluate the effects of aging on UG-knockout mice, 16/16 animals developed malignant tumors, whereas the wild-type littermates (n = 25) remained apparently healthy even after 1½ years. In the present investigation, we sought to determine the effects of induced-expression of hUG in human cancer cells by transfecting several cell lines derived from adenocarcinomas of various organs with an hUG-cDNA construct. We demonstrate that induced hUG expression reverses at least two of the most important characteristics of the transformed phenotype (i.e., anchorage-independent growth on soft agar and extracellular matrix invasion) of only those cancer cells that also express the hUG receptor. Similarly, treatment of the nontransfected, receptor-positive adenocarcinoma cells with purified recombinant hUG yielded identical results. Taken together, these data define receptor-mediated, autocrine and paracrine pathways through which hUG reverses the transformed phenotype of cancer cells and consequently, may have tumor suppressor-like effects.

The lethal mutation of the mouse wasted (wst) is a deletion that abolishes expression of a tissue-specific isoform of translation elongation factor 1α, encoded by the Eef1a2 gene

We have identified the mutation responsible for the autosomal recessive wasted (wst) mutation of the mouse. Wasted mice are characterized by wasting and neurological and immunological abnormalities starting at 21 days after birth; they die by 28 days. A deletion of 15.8 kb in wasted mice abolishes expression of a gene called Eef1a2, encoding a protein that is 92% identical at the amino acid level to the translation elongation factor EF1α (locus Eef1a). We have found no evidence for the involvement of another gene in this deletion. Expression of Eef1a2 is reciprocal with that of Eef1a. Expression of Eef1a2 takes over from Eef1a in heart and muscle at precisely the time at which the wasted phenotype becomes manifest. These data suggest that there are tissue-specific forms of the translation elongation apparatus essential for postnatal survival in the mouse.

Identification and characterization of the ARP1 gene, a target for the human acute leukemia ALL1 gene

ALL1, the human homologue of Drosophila trithorax, is directly involved in human acute leukemias associated with abnormalities at 11q23. Using the differential display method, we isolated a gene that is down-regulated in All1 double-knockout mouse embryonic stem (ES) cells. The gene, designated ARP1 (also termed RIEG, Ptx2, or Otlx2), is a member of a family of homeotic genes containing a short motif shared with several homeobox genes. Using a bacterially synthesized All1 polypeptide encompassing the AT-hook motifs, we identified a 0.5-kb ARP1 DNA fragment that preferentially bound to the polypeptide. Within this DNA, a region of ≈100 bp was protected by the polypeptide from digestion with ExoIII and DNase I. Whole-mount in situ hybridization to early mouse embryos of 9.5–10.5 days indicated a complex pattern of Arp1 expression spatially overlapping with the expression of All1. Although the ARP1 gene is expressed strongly in bone marrow cells, no transcripts were detected in six leukemia cell lines with 11q23 translocations. These results suggest that ARP1 is up-regulated by the All1 protein, possibly through direct interaction with an upstream DNA sequence of the former. The results are also consistent with the suggestion that ALL1 chimeric proteins resulting from 11q23 abnormalities act in a dominant negative fashion.

A third member of the synapsin gene family

Synapsins are a family of neuron-specific synaptic vesicle-associated phosphoproteins that have been implicated in synaptogenesis and in the modulation of neurotransmitter release. In mammals, distinct genes for synapsins I and II have been identified, each of which gives rise to two alternatively spliced isoforms. We have now cloned and characterized a third member of the synapsin gene family, synapsin III, from human DNA. Synapsin III gives rise to at least one protein isoform, designated synapsin IIIa, in several mammalian species. Synapsin IIIa is associated with synaptic vesicles, and its expression appears to be neuron-specific. The primary structure of synapsin IIIa conforms to the domain model previously described for the synapsin family, with domains A, C, and E exhibiting the highest degree of conservation. Synapsin IIIa contains a novel domain, termed domain J, located between domains C and E. The similarities among synapsins I, II, and III in domain organization, neuron-specific expression, and subcellular localization suggest a possible role for synapsin III in the regulation of neurotransmitter release and synaptogenesis. The human synapsin III gene is located on chromosome 22q12–13, which has been identified as a possible schizophrenia susceptibility locus. On the basis of this localization and the well established neurobiological roles of the synapsins, synapsin III represents a candidate gene for schizophrenia.

Amyrel, a paralogous gene of the amylase gene family in Drosophila melanogaster and the Sophophora subgenus

We describe a gene from Drosophila melanogaster related to the alpha-amylase gene Amy. This gene, which exists as a single copy, was named Amyrel. It is strikingly divergent from Amy because the amino acid divergence is 40%. The coding sequence is interrupted by a short intron at position 655, which is unusual in amylase genes. Amyrel has also been cloned in Drosophila ananassae, Drosophila pseudoobscura, and Drosophila subobscura and is likely to be present throughout the Sophophora subgenus, but, to our knowledge, it has not been detected outside. Unexpectedly, there is a strong conservation of 5′ and 3′ flanking regions between Amyrel genes from different species, which is not the case for Amy and which suggests that selection acts on these regions. In contrast to the Amy genes, Amyrel is transcribed in larvae of D. melanogaster but not in adults. However, the protein has not been detected yet. Amyrel evolves about twice as fast as Amy in the several species studied. We suggest that this gene could result from a duplication of Amy followed by accelerated and selected divergence toward a new adaptation.

Characterization of mice deficient in aromatase (ArKO) because of targeted disruption of the cyp19 gene

The formation of estrogens from C19 steroids is catalyzed by aromatase cytochrome P450 (P450arom), the product of the cyp19 gene. The actions of estrogen include dimorphic anatomical, functional, and behavioral effects on the development of both males and females, considerations that prompted us to examine the consequences of deficiency of aromatase activity in mice. Mice lacking a functional aromatase enzyme (ArKO) were generated by targeted disruption of the cyp19 gene. Male and female ArKO mice were born with the expected Mendelian frequency from F1 parents and grew to adulthood. Female ArKO mice at 9 weeks of age displayed underdeveloped external genitalia and uteri. Ovaries contained numerous follicles with abundant granulosa cells and evidence of antrum formation that appeared arrested before ovulation. No corpora lutea were present. Additionally the stroma were hyperplastic with structures that appeared to be atretic follicles. Development of the mammary glands approximated that of a prepubertal female. Examination of male ArKO mice of the same age revealed essentially normal internal anatomy but with enlargement of the male accessory sex glands because of increased content of secreted material. The testes appeared normal. Male ArKO mice are capable of breeding and produce litters of approximately average size. Whereas serum estradiol levels were at the limit of detection, testosterone levels were elevated, as were the levels of follicle-stimulating hormone and luteinizing hormone. The phenotype of these animals differs markedly from that of the previously reported ERKO mice, in which the estrogen receptor α is deleted by targeted disruption.

Mutation in the tau gene in familial multiple system tauopathy with presenile dementia

Familial multiple system tauopathy with presenile dementia (MSTD) is a neurodegenerative disease with an abundant filamentous tau protein pathology. It belongs to the group of familial frontotemporal dementias with Parkinsonism linked to chromosome 17 (FTDP-17), a major class of inherited dementing disorders whose genetic basis is unknown. We now report a G to A transition in the intron following exon 10 of the gene for microtubule-associated protein tau in familial MSTD. The mutation is located at the 3′ neighboring nucleotide of the GT splice-donor site and disrupts a predicted stem-loop structure. We also report an abnormal preponderance of soluble tau protein isoforms with four microtubule-binding repeats over isoforms with three repeats in familial MSTD. This most likely accounts for our previous finding that sarkosyl-insoluble tau protein extracted from the filamentous deposits in familial MSTD consists only of tau isoforms with four repeats. These findings reveal that a departure from the normal ratio of four-repeat to three-repeat tau isoforms leads to the formation of abnormal tau filaments. The results show that dysregulation of tau protein production can cause neurodegeneration and imply that the FTDP-17 gene is the tau gene. This work has major implications for Alzheimer’s disease and other tauopathies.

Isolation and characterization of mammalian homologs of the Drosophila gene glial cells missing

The glial cells missing (gcm) gene in Drosophila encodes a transcription factor that determines the choice between glial and neuronal fates. We report here the isolation of two mammalian gcm homologs, Gcm1 and Gcm2, and the characterization of their expression patterns during embryonic development. Although Gcm2 is expressed in neural tissues at a low level, the major sites of expression for both of the mammalian genes are nonneural, suggesting that the functions of the mammalian homologs have diverged and diversified. However, when expressed ectopically, Gcm1 can substitute functionally for Drosophila gcm by transforming presumptive neurons into glia. Thus, certain biochemical properties, although not the specificity of the tissue in which the gene is expressed, have been conserved through the evolution of the Gcm gene family.

In situ detection of the hypermethylation-induced inactivation of the p16 gene as an early event in oncogenesis

We have developed a technique, methylation-specific PCR in situ hybridization (MSP-ISH), which allows for the methylation status of specific DNA sequences to be visualized in individual cells. We use MSP-ISH to monitor the timing and consequences of aberrant hypermethylation of the p16 tumor suppresser gene during the progression of cancers of the lung and cervix. Hypermethylation of p16 was localized only to the neoplastic cells in both in situ lesions and invasive cancers, and was associated with loss of p16 protein expression. MSP-ISH allowed us to dissect the surprising finding that p16 hypermethylation occurs in cervical carcinoma. This tumor is associated with infection of the oncogenic human papillomavirus, which expresses a protein, E7, that inactivates the retinoblastoma (Rb) protein. Thus, simultaneous Rb and p16 inactivation would not be needed to abrogate the critical cyclin D–Rb pathway. MSP-ISH reveals that p16 hypermethylation occurs heterogeneously within early cervical tumor cell populations that are separate from those expressing viral E7 transcripts. In advanced cervical cancers, the majority of cells have a hypermethylated p16, lack p16 protein, but no longer express E7. These data suggest that p16 inactivation is selected as the most effective mechanism of blocking the cyclin D–Rb pathway during the evolution of an invasive cancer from precursor lesions. These studies demonstrate that MSP-ISH is a powerful approach for studying the dynamics of aberrant methylation of critical tumor suppressor genes during tumor evolution.

Transcription initiation is controlled by three core promoter elements in the hgl5 gene of the protozoan parasite Entamoeba histolytica

Entamoeba histolytica is a single cell eukaryote that is the etiologic agent of amoebic colitis. Core promoter elements of E. histolytica protein encoding genes include a TATA-like sequence (GTATTTAAAG/C) at −30, a novel element designated GAAC (GAACT) that has a variable location between TATA and the site of transcription initiation, and a putative initiator (Inr) element (AAAAATTCA) overlying the site of transcription initiation. The presence of three separate conserved sequences in a eukaryotic core promoter is unprecedented and prompted examination of their roles in regulating transcription initiation. Alterations of all three regions in the hgl5 gene decreased reporter gene activity with the greatest effect seen by mutation of the GAAC element. Positional analysis of the TATA box demonstrated that transcription initiated consistently 30–31 bases downstream of the TATA region. Mutation of either the TATA or GAAC elements resulted in the appearance of new transcription start sites upstream of +1 in the promoter of the hgl5 gene. Mutation of the Inr element resulted in no change in the site of transcription initiation; however, in the presence of a mutated TATA and GAAC regions, the Inr element controlled the site of transcription initiation. We conclude that all three elements play a role in determining the site of transcription initiation. The variable position of the GAAC element relative to the site of transcription initiation, and the multiple transcription initiations that resulted from its mutation, indicate that the GAAC element has an important and apparently novel role in transcriptional control in E. histolytica.

Mitochondrial DNA rearrangements of Podospora anserina are under the control of the nuclear gene grisea

Podospora anserina is a filamentous fungus with a limited life span. Life span is controlled by nuclear and extranuclear genetic traits. Herein we report the nature of four alterations in the nuclear gene grisea that lead to an altered morphology, a defect in the formation of female gametangia, and an increased life span. Three sequence changes are located in the 5′ upstream region of the grisea ORF. One mutation is a G → A transition at the 5′ splice site of the single intron of the gene, leading to a RNA splicing defect. This loss-of-function affects the amplification of the first intron of the mitochondrial cytochrome c oxidase subunit I gene (COI) and the specific mitochondrial DNA rearrangements that occur during senescence of wild-type strains. Our results indicate that the nuclear gene grisea is part of a molecular machinery involved in the control of mitochondrial DNA reorganizations. These DNA instabilities accelerate but are not a prerequisite for the aging of P. anserina cultures.

Proximo–distal specification in the wing disc of Drosophila by the nubbin gene

Mutations in the nubbin (nub) gene have a phenotype consisting of a severe wing size reduction and pattern alterations, such as transformations of distal elements into proximal ones. nub expression is restricted to the wing pouch cells in wing discs since early larval development. These effects are also observed in genetic mosaics where cell proliferation is reduced in all wing blade regions autonomously, and transformation into proximal elements is observed in distal clones. Clones located in the proximal region of the wing blade cause in addition nonautonomous reduction of the whole wing. Cell lineage experiments in a nub mutant background show that clones respect neither the anterior–posterior nor the dorsal–ventral boundary but that the selector genes have been correctly expressed since early larval development. The phenotypes of nub el and nub dpp genetic combinations are synergistic and the overexpression of dpp in clones in nub wings does not result in overproliferation of the surrounding wild-type cells. We discuss the role of nub in the wing’s proximo–distal axis and in the formation of compartment boundaries.