A minimal gene set for cellular life derived by comparison of complete bacterial genomes.

The recently sequenced genome of the parasitic bacterium Mycoplasma genitalium contains only 468 identified protein-coding genes that have been dubbed a minimal gene complement [Fraser, C.M., Gocayne, J.D., White, O., Adams, M.D., Clayton, R.A., et al. (1995) Science 270, 397-403]. Although the M. genitalium gene complement is indeed the smallest among known cellular life forms, there is no evidence that it is the minimal self-sufficient gene set. To derive such a set, we compared the 468 predicted M. genitalium protein sequences with the 1703 protein sequences encoded by the other completely sequenced small bacterial genome, that of Haemophilus influenzae. M. genitalium and H. influenzae belong to two ancient bacterial lineages, i.e., Gram-positive and Gram-negative bacteria, respectively. Therefore, the genes that are conserved in these two bacteria are almost certainly essential for cellular function. It is this category of genes that is most likely to approximate the minimal gene set. We found that 240 M. genitalium genes have orthologs among the genes of H. influenzae. This collection of genes falls short of comprising the minimal set as some enzymes responsible for intermediate steps in essential pathways are missing. The apparent reason for this is the phenomenon that we call nonorthologous gene displacement when the same function is fulfilled by nonorthologous proteins in two organisms. We identified 22 nonorthologous displacements and supplemented the set of orthologs with the respective M. genitalium genes. After examining the resulting list of 262 genes for possible functional redundancy and for the presence of apparently parasite-specific genes, 6 genes were removed. We suggest that the remaining 256 genes are close to the minimal gene set that is necessary and sufficient to sustain the existence of a modern-type cell. Most of the proteins encoded by the genes from the minimal set have eukaryotic or archaeal homologs but seven key proteins of DNA replication do not. We speculate that the last common ancestor of the three primary kingdoms had an RNA genome. Possibilities are explored to further reduce the minimal set to model a primitive cell that might have existed at a very early stage of life evolution.

Inhibition of DNA methyltransferase stimulates the expression of signal transducer and activator of transcription 1, 2, and 3 genes in colon tumor cells

Inhibitors of DNA methyltransferase, typified by 5-aza-2′-deoxycytidine (5-Aza-CdR), induce the expression of genes transcriptionally down-regulated by de novo methylation in tumor cells. We utilized gene expression microarrays to examine the effects of 5-Aza-CdR treatment in HT29 colon adenocarcinoma cells. This analysis revealed the induction of a set of genes that implicated IFN signaling in the HT29 cellular response to 5-Aza-CdR. Subsequent investigations revealed that the induction of this gene set correlates with the induction of signal transducer and activator of transcription (STAT) 1, 2, and 3 genes and their activation by endogenous IFN-α. These observations implicate the induction of the IFN-response pathway as a major cellular response to 5-Aza-CdR and suggests that the expression of STATs 1, 2, and 3 can be regulated by DNA methylation. Consistent with STAT’s limiting cell responsiveness to IFN, we found that 5-Aza-CdR treatment sensitized HT29 cells to growth inhibition by exogenous IFN-α2a, indicating that 5-Aza-CdR should be investigated as a potentiator of IFN responsiveness in certain IFN-resistant tumors.

The chromosomal distribution of mouse odorant receptor genes.

Odorant receptors (ORs) on nasal olfactory sensory neurons are encoded by a large multigene family. Each member of the family is expressed in a small percentage of neurons that are confined to one of several spatial zones in the nose but are randomly distributed throughout that zone. This pattern of expression suggests that when the sensory neuron selects which OR gene to express it may be confined to a particular zonal gene set of several hundred OR genes but select from among the members of that set via a stochastic mechanism. Both locus-dependent and locus-independent models of OR gene choice have been proposed. To investigate the feasibility of these models, we determined the chromosomal locations of 21 OR genes expressed in four different spatial zones. We found that OR genes are clustered within multiple loci that are broadly distributed in the genome. These loci lie within paralogous chromosomal regions that appear to have arisen by duplications of large chromosomal domains followed by extensive gene duplication and divergence. Our studies show that OR genes expressed in the same zone map to numerous loci; moreover, a single locus can contain genes expressed in different zones. These findings raise the possibility that OR gene choice may be locus-independent or involve consecutive stochastic choices.

Control of fertilization-independent endosperm development by the MEDEA polycomb gene in Arabidopsis

Higher plant reproduction is unique because two cells are fertilized in the haploid female gametophyte. Egg and sperm nuclei fuse to form the embryo. A second sperm nucleus fuses with the central cell nucleus that replicates to generate the endosperm, a tissue that supports embryo development. To understand mechanisms that initiate reproduction, we isolated a mutation in Arabidopsis, f644, that allows for replication of the central cell and subsequent endosperm development without fertilization. When mutant f644 egg and central cells are fertilized by wild-type sperm, embryo development is inhibited, and endosperm is overproduced. By using a map-based strategy, we cloned and sequenced the F644 gene and showed that it encodes a SET-domain polycomb protein. Subsequently, we found that F644 is identical to MEDEA (MEA), a gene whose maternal-derived allele is required for embryogenesis [Grossniklaus, U., Vielle-Calzada, J.-P., Hoeppner, M. A. & Gagliano, W. B. (1998) Science 280, 446–450]. Together, these results reveal functions for plant polycomb proteins in the suppression of central cell proliferation and endosperm development. We discuss models to explain how polycomb proteins function to suppress endosperm and promote embryo development.

The C-terminal SET domains of ALL-1 and TRITHORAX interact with the INI1 and SNR1 proteins, components of the SWI/SNF complex

The ALL-1 gene was discovered by virtue of its involvement in human acute leukemia. Its Drosophila homolog trithorax (trx) is a member of the trx-Polycomb gene family, which maintains correct spatial expression of the Antennapedia and bithorax complexes during embryogenesis. The C-terminal SET domain of ALL-1 and TRITHORAX (TRX) is a 150-aa motif, highly conserved during evolution. We performed yeast two hybrid screening of Drosophila cDNA library and detected interaction between a TRX polypeptide spanning SET and the SNR1 protein. SNR1 is a product of snr1, which is classified as a trx group gene. We found parallel interaction in yeast between the SET domain of ALL-1 and the human homolog of SNR1, INI1 (hSNF5). These results were confirmed by in vitro binding studies and by demonstrating coimmunoprecipitation of the proteins from cultured cells and/or transgenic flies. Epitope-tagged SNR1 was detected at discrete sites on larval salivary gland polytene chromosomes, and these sites colocalized with around one-half of TRX binding sites. Because SNR1 and INI1 are constituents of the SWI/SNF complex, which acts to remodel chromatin and consequently to activate transcription, the interactions we observed suggest a mechanism by which the SWI/SNF complex is recruited to ALL-1/trx targets through physical interactions between the C-terminal domains of ALL-1 and TRX and INI1/SNR1.

Recessive and dominant mutations in the ethylene biosynthetic gene ACS5 of Arabidopsis confer cytokinin insensitivity and ethylene overproduction, respectively

We identified a set of cytokinin-insensitive mutants by using a screen based on the ethylene-mediated triple response observed after treatment with low levels of cytokinins. One group of these mutants disrupts ACS5, a member of the Arabidopsis gene family that encodes 1-aminocyclopropane-1-carboxylate synthase, the first enzyme in ethylene biosynthesis. The ACS5 isoform is mainly responsible for the sustained rise in ethylene biosynthesis observed in response to low levels of cytokinin and appears to be regulated primarily by a posttranscriptional mechanism. Furthermore, the dominant ethylene-overproducing mutant eto2 was found to be the result of an alteration of the carboxy terminus of ACS5, suggesting that this domain acts as a negative regulator of ACS5 function.

The gastrulation defective gene of Drosophila melanogaster is a member of the serine protease superfamily

The establishment of dorsal–ventral polarity in the oocyte involves two sets of genes. One set belongs to the gurken-torpedo signaling pathway and affects the development of the egg chorion as well as the polarity of the embryo. The second set of genes affects only the dorsal–ventral polarity of the embryo but not the eggshell. gastrulation defective is one of the earliest acting of this second set of maternally required genes. We have cloned and characterized the gastrulation defective gene and determined that it encodes a protein structurally related to the serine protease superfamily, which also includes the Snake, Easter, and Nudel proteins. These data provide additional support for the involvement of a protease cascade in generating an asymmetric signal (i.e., asymmetric Spätzle activity) during establishment of dorsal–ventral polarity in the Drosophila embryo.

A gene cluster for macrolide antibiotic biosynthesis in Streptomyces venezuelae: Architecture of metabolic diversity

In a survey of microbial systems capable of generating unusual metabolite structural variability, Streptomyces venezuelae ATCC 15439 is notable in its ability to produce two distinct groups of macrolide antibiotics. Methymycin and neomethymycin are derived from the 12-membered ring macrolactone 10-deoxymethynolide, whereas narbomycin and pikromycin are derived from the 14-membered ring macrolactone, narbonolide. This report describes the cloning and characterization of the biosynthetic gene cluster for these antibiotics. Central to the cluster is a polyketide synthase locus (pikA) that encodes a six-module system comprised of four multifunctional proteins, in addition to a type II thioesterase (TEII). Immediately downstream is a set of genes for desosamine biosynthesis (des) and macrolide ring hydroxylation. The study suggests that Pik TEII plays a role in forming a metabolic branch through which polyketides of different chain length are generated, and the glycosyl transferase (encoded by desVII) has the ability to catalyze glycosylation of both the 12- and 14-membered ring macrolactones. Moreover, the pikC-encoded P450 hydroxylase provides yet another layer of structural variability by introducing regiochemical diversity into the macrolide ring systems. The data support the notion that the architecture of the pik gene cluster as well as the unusual substrate specificity of particular enzymes contributes to its ability to generate four macrolide antibiotics.

Expression of the Hox gene complex in the indirect development of a sea urchin

Hox complex genes control spatial patterning mechanisms in the development of arthropod and vertebrate body plans. Hox genes are all expressed during embryogenesis in these groups, which are all directly developing organisms in that embryogenesis leads at once to formation of major elements of the respective adult body plans. In the maximally indirect development of a large variety of invertebrates, the process of embryogenesis leads only to a free-living, bilaterally organized feeding larva. Maximal indirect development is exemplified in sea urchins. The 5-fold radially symmetric adult body plan of the sea urchin is generated long after embryogenesis is complete, by a separate process occurring within imaginal tissues set aside in the larva. The single Hox gene complex of Strongylocentrotus purpuratus contains 10 genes, and expression of eight of these genes was measured by quantitative methods during both embryonic and larval developmental stages and also in adult tissues. Only two of these genes are used significantly during the entire process of embryogenesis per se, although all are copiously expressed during the stages when the adult body plan is forming in the imaginal rudiment. They are also all expressed in various combinations in adult tissues. Thus, development of a microscopic, free-living organism of bilaterian grade, the larva, does not appear to require expression of the Hox gene cluster as such, whereas development of the adult body plan does. These observations reflect on mechanisms by which bilaterian metazoans might have arisen in Precambrian evolution.

Functioning of the Drosophila integral U1/U2 protein Snf independent of U1 and U2 small nuclear ribonucleoprotein particles is revealed by snf+ gene dose effects

Snf, encoded by sans fille, is the Drosophila homolog of mammalian U1A and U2B′′ and is an integral component of U1 and U2 small nuclear ribonucleoprotein particles (snRNPs). Surprisingly, changes in the level of this housekeeping protein can specifically affect autoregulatory activity of the RNA-binding protein Sex-lethal (Sxl) in an action that we infer must be physically separate from Snf’s functioning within snRNPs. Sxl is a master switch gene that controls its own pre-mRNA splicing as well as splicing for subordinate switch genes that regulate sex determination and dosage compensation. Exploiting an unusual new set of mutant Sxl alleles in an in vivo assay, we show that Snf is rate-limiting for Sxl autoregulation when Sxl levels are low. In such situations, increasing either maternal or zygotic snf+ dose enhances the positive autoregulatory activity of Sxl for Sxl somatic pre-mRNA splicing without affecting Sxl activities toward its other RNA targets. In contrast, increasing the dose of genes encoding either the integral U1 snRNP protein U1-70k, or the integral U2 snRNP protein SF3a60, has no effect. Increased snf+ enhances Sxl autoregulation even when U1-70k and SF3a60 are reduced by mutation to levels that, in the case of SF3a60, demonstrably interfere with Sxl autoregulation. The observation that increased snf+ does not suppress other phenotypes associated with mutations that reduce U1-70k or SF3a60 is additional evidence that snf+ dose effects are not caused by increased snRNP levels. Mammalian U1A protein, like Snf, has a snRNP-independent function.

Toward controlling gene expression at will: Specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks

To create a universal system for the control of gene expression, we have studied methods for the construction of novel polydactyl zinc finger proteins that recognize extended DNA sequences. Elsewhere we have described the generation of zinc finger domains recognizing sequences of the 5′-GNN-3′ subset of a 64-member zinc finger alphabet. Here we report on the use of these domains as modular building blocks for the construction of polydactyl proteins specifically recognizing 9- or 18-bp sequences. A rapid PCR assembly method was developed that, together with this predefined set of zinc finger domains, provides ready access to 17 million novel proteins that bind the 5′-(GNN)6-3′ family of 18-bp DNA sites. To examine the efficacy of this strategy in gene control, the human erbB-2 gene was chosen as a model. A polydactyl protein specifically recognizing an 18-bp sequence in the 5′-untranslated region of this gene was converted into a transcriptional repressor by fusion with Krüppel-associated box (KRAB), ERD, or SID repressor domains. Transcriptional activators were generated by fusion with the herpes simplex VP16 activation domain or with a tetrameric repeat of VP16’s minimal activation domain, termed VP64. We demonstrate that both gene repression and activation can be achieved by targeting designed proteins to a single site within the transcribed region of a gene. We anticipate that gene-specific transcriptional regulators of the type described here will find diverse applications in gene therapy, functional genomics, and the generation of transgenic organisms.

Structural assignments to the Mycoplasma genitalium proteins show extensive gene duplications and domain rearrangements

The parasitic bacterium Mycoplasma genitalium has a small, reduced genome with close to a basic set of genes. As a first step toward determining the families of protein domains that form the products of these genes, we have used the multiple sequence programs psi-blast and geanfammer to match the sequences of the 467 gene products of M. genitalium to the sequences of the domains that form proteins of known structure [Protein Data Bank (PDB) sequences]. PDB sequences (274) match all of 106 M. genitalium sequences and some parts of another 85; thus, 41% of its total sequences are matched in all or part. The evolutionary relationships of the PDB domains that match M. genitalium are described in the structural classification of proteins (SCOP) database. Using this information, we show that the domains in the matched M. genitalium sequences come from 114 superfamilies and that 58% of them have arisen by gene duplication. This level of duplication is more than twice that found by using pairwise sequence comparisons. The PDB domain matches also describe the domain structure of the matched sequences: just over a quarter contain one domain and the rest have combinations of two or more domains.

Gene dosage and stochastic effects determine the severity and direction of uniparental ribosomal RNA gene silencing (nucleolar dominance) in Arabidopsis allopolyploids

Nucleolar dominance is an epigenetic phenomenon in which one parental set of ribosomal RNA (rRNA) genes is silenced in an interspecific hybrid. In natural Arabidopsis suecica, an allotetraploid (amphidiploid) hybrid of Arabidopsis thaliana and Cardaminopsis arenosa, the A. thaliana rRNA genes are repressed. Interestingly, A. thaliana rRNA gene silencing is variable in synthetic Arabidopsis suecica F1 hybrids. Two generations are needed for A. thaliana rRNA genes to be silenced in all lines, revealing a species-biased direction but stochastic onset to nucleolar dominance. Backcrossing synthetic A. suecica to tetraploid A. thaliana yielded progeny with active A. thaliana rRNA genes and, in some cases, silenced C. arenosa rRNA genes, showing that the direction of dominance can be switched. The hypothesis that naturally dominant rRNA genes have a superior binding affinity for a limiting transcription factor is inconsistent with dominance switching. Inactivation of a species-specific transcription factor is argued against by showing that A. thaliana and C. arenosa rRNA genes can be expressed transiently in the other species. Transfected A. thaliana genes are also active in A. suecica protoplasts in which chromosomal A. thaliana genes are repressed. Collectively, these data suggest that nucleolar dominance is a chromosomal phenomenon that results in coordinate or cooperative silencing of rRNA genes.

An Intronic Enhancer Is Required for Deflagellation-induced Transcriptional Regulation of a Chlamydomonas reinhardtii Dynein Gene

Chlamydomonas reinhardtii flagellar regeneration is accompanied by rapid induction of genes encoding a large set of flagellar structural components and provides a model system to study coordinate gene regulation and organelle assembly. After deflagellation, the abundance of a 70-kDa flagellar dynein intermediate chain (IC70, encoded by ODA6) mRNA increases approximately fourfold within 40 min and returns to predeflagellation levels by ∼90 min. We show by nuclear run-on that this increase results, in part, from increased rates of transcription. To localize cis induction elements, we created an IC70 minigene and measured accumulation, in C. reinhardtii, of transcripts from the endogenous gene and from introduced promoter deletion constructs. Clones containing 416 base pairs (bp) of 5′- and 2 kilobases (kb) of 3′-flanking region retained all sequences necessary for a normal pattern of mRNA abundance change after deflagellation. Extensive 5′- and 3′- flanking region deletions, which removed multiple copies of a proposed deflagellation-response element (the tub box), did not eliminate induction, and the IC70 5′-flanking region alone did not confer deflagellation responsiveness to a promoterless arylsulfatase (ARS) gene. Instead, an intron in the IC70 gene 5′-untranslated region was found to contain the deflagellation response element. These results suggest that the tub box does not play an essential role in deflagellation-induced transcriptional regulation of this dynein gene.

A Novel Yeast Screen for Mitotic Arrest Mutants Identifies DOC1, a New Gene Involved in Cyclin Proteolysis

B-type cyclins are rapidly degraded at the transition between metaphase and anaphase and their ubiquitin-mediated proteolysis is required for cells to exit mitosis. We used a novel enrichment to isolate new budding mutants that arrest the cell cycle in mitosis. Most of these mutants lie in the CDC16, CDC23, and CDC27 genes, which have already been shown to play a role in cyclin proteolysis and encode components of a 20S complex (called the cyclosome or anaphase promoting complex) that ubiquitinates mitotic cyclins. We show that mutations in CDC26 and a novel gene, DOC1, also prevent mitotic cyclin proteolysis. Mutants in either gene arrest as large budded cells with high levels of the major mitotic cyclin (Clb2) protein at 37°C and cannot degrade Clb2 in G1-arrested cells. Cdc26 associates in vivo with Doc1, Cdc16, Cdc23, and Cdc27. In addition, the majority of Doc1 cosediments at 20S with Cdc27 in a sucrose gradient, indicating that Cdc26 and Doc1 are components of the anaphase promoting complex.