Identification of 10 novel snoRNA gene clusters from Arabidopsis thaliana

Ten novel small nucleolar RNA (snoRNA) gene clusters, consisting of two or three snoRNA genes, respectively, were identified from Arabidopsis thaliana. Twelve of the 25 snoRNA genes in these clusters are homologous to those of yeast and mammals according to the conserved antisense sequences that guide 2′-O-ribose methylation of rRNA. The remaining 13 snoRNA genes, including two 5.8S rRNA methylation guides, are new genes identified from A.thaliana. Interestingly, seven methylated nucleotides, predicted by novel snoRNAs Z41a–Z46, are methylated neither in yeast nor in vertebrates. Using primer extension at low dNTP concentration the six methylation sites were determined as expected. These snoRNAs were recognized as specific guides for 2′-O-ribose methylation of plant rRNAs. Z42, however, did not guide the expected methylation of 25S rRNA in our assay. Thus, its function remains to be elucidated. The intergenic spacers of the gene clusters are rich in uridine (up to 40%) and most of them range in size from 35 to 100 nt. Lack of a conserved promoter element in each spacer and the determination of polycistronic transcription from a cluster by RT–PCR assay suggest that the snoRNAs encoded in the clusters are transcribed as a polycistron under an upstream promoter, and individual snoRNAs are released after processing of the precursor. Numerous snoRNA gene clusters identified from A.thaliana and other organisms suggest that the snoRNA gene cluster is an ancient gene organization existing abundantly in plants.

The chaperone/usher pathways of Pseudomonas aeruginosa: Identification of fimbrial gene clusters (cup) and their involvement in biofilm formation

Pseudomonas aeruginosa, an important opportunistic human pathogen, persists in certain tissues in the form of specialized bacterial communities, referred to as biofilm. The biofilm is formed through series of interactions between cells and adherence to surfaces, resulting in an organized structure. By screening a library of Tn5 insertions in a nonpiliated P. aeruginosa strain, we identified genes involved in early stages of biofilm formation. One class of mutations identified in this study mapped in a cluster of genes specifying the components of a chaperone/usher pathway that is involved in assembly of fimbrial subunits in other microorganisms. These genes, not previously described in P. aeruginosa, were named cupA1–A5. Additional chaperone/usher systems (CupB and CupC) have been also identified in the genome of P. aeruginosa PAO1; however, they do not appear to play a role in adhesion under the conditions where the CupA system is expressed and functions in surface adherence. The identification of these putative adhesins on the cell surface of P. aeruginosa suggests that this organism possess a wide range of factors that function in biofilm formation. These structures appear to be differentially regulated and may function at distinct stages of biofilm formation, or in specific environments colonized by this organism.

In vitro reconstruction of the Aspergillus (= Emericella) nidulans genome

A physical map of the 31-megabase Aspergillus nidulans genome is reported, in which 94% of 5,134 cosmids are assigned to 49 contiguous segments. The physical map is the result of a two-way ordering process, in which clones and probes were ordered simultaneously on a binary DNA/DNA hybridization matrix. Compression by elimination of redundant clones resulted in a minimal map, which is a chromosome walk. Repetitive DNA is nonrandomly dispersed in the A. nidulans genome, reminiscent of heterochromatic banding patterns of higher eukaryotes. We hypothesize gene clusters may arise by horizontal transfer and spread by transposition to explain the nonrandom pattern of repeats along chromosomes.

Coiled Bodies and U2 snRNA Genes Adjacent to Coiled Bodies Are Enriched in Factors Required for snRNA Transcription

A significant percentage of the gene clusters that contain the human genes for U1 small nuclear RNA (snRNA) or for U2 snRNA have been found associated with small nuclear domains, known as coiled bodies. We show here, by immunofluorescent labeling of human cells, that coiled bodies are enriched in factors required for the transcription of these snRNA genes. The 45-kDa γ-subunit of the transcription factor, proximal element sequence-binding transcription factor (PTF), which is specific for the snRNA genes, was found in high concentrations in coiled bodies, along with the general transcription factor TATA-box binding protein and a subset of RNA polymerase II. We show that the transcription factors and RNA polymerase II are concentrated in irregularly shaped domains that not only overlap with coiled bodies but also extend to their immediate surroundings. Fluorescent in situ hybridization showed that these domains can overlap with U2 snRNA genes adjacent to coiled bodies. In addition, we found the domains to contain newly synthesized RNA, visualized by 5-bromo-uridine triphosphate labeling. Our data suggest that coiled bodies are involved in the expression of snRNA genes, which leads us to propose the model that coiled bodies are associated with snRNA genes to facilitate and regulate their transcription. These findings point to a general principle of higher order organization of gene expression in the nucleus.

Nuclear Domains Enriched in RNA 3′-processing Factors Associate with Coiled Bodies and Histone Genes in a Cell Cycle–dependent Manner

Nuclear domains, called cleavage bodies, are enriched in the RNA 3′-processing factors CstF 64 kDa and and CPSF 100 kDa. Cleavage bodies have been found either overlapping with or adjacent to coiled bodies. To determine whether the spatial relationship between cleavage bodies and coiled bodies was influenced by the cell cycle, we performed cell synchronization studies. We found that in G1 phase cleavage bodies and coiled bodies were predominantly coincident, whereas in S phase they were mostly adjacent to each other. In G2 cleavage bodies were often less defined or absent, suggesting that they disassemble at this point in the cell cycle. A small number of genetic loci have been reported to be juxtaposed to coiled bodies, including the genes for U1 and U2 small nuclear RNA as well as the two major histone gene clusters. Here we show that cleavage bodies do not overlap with small nuclear RNA genes but do colocalize with the histone genes next to coiled bodies. These findings demonstrate that the association of cleavage bodies and coiled bodies is both dynamic and tightly regulated and suggest that the interaction between these nuclear neighbors is related to the cell cycle–dependent expression of histone genes.

Hox cluster genomics in the horn shark, Heterodontus francisci

Reconstructing the evolutionary history of Hox cluster origins will lead to insights into the developmental and evolutionary significance of Hox gene clusters in vertebrate phylogeny and to their role in the origins of various vertebrate body plans. We have isolated two Hox clusters from the horn shark, Heterodontus francisci. These have been sequenced and compared with one another and with other chordate Hox clusters. The results show that one of the horn shark clusters (HoxM) is orthologous to the mammalian HoxA cluster and shows a structural similarity to the amphioxus cluster, whereas the other shark cluster (HoxN) is orthologous to the mammalian HoxD cluster based on cluster organization and a comparison with noncoding and Hox gene-coding sequences. The persistence of an identifiable HoxA cluster over an 800-million-year divergence time demonstrates that the Hox gene clusters are highly integrated and structured genetic entities. The data presented herein identify many noncoding sequence motifs conserved over 800 million years that may function as genetic control motifs essential to the developmental process.

Grass genomes

For the most part, studies of grass genome structure have been limited to the generation of whole-genome genetic maps or the fine structure and sequence analysis of single genes or gene clusters. We have investigated large contiguous segments of the genomes of maize, sorghum, and rice, primarily focusing on intergenic spaces. Our data indicate that much (>50%) of the maize genome is composed of interspersed repetitive DNAs, primarily nested retrotransposons that insert between genes. These retroelements are less abundant in smaller genome plants, including rice and sorghum. Although 5- to 200-kb blocks of methylated, presumably heterochromatic, retrotransposons flank most maize genes, rice and sorghum genes are often adjacent. Similar genes are commonly found in the same relative chromosomal locations and orientations in each of these three species, although there are numerous exceptions to this collinearity (i.e., rearrangements) that can be detected at the levels of both the recombinational map and cloned DNA. Evolutionarily conserved sequences are largely confined to genes and their regulatory elements. Our results indicate that a knowledge of grass genome structure will be a useful tool for gene discovery and isolation, but the general rules and biological significance of grass genome organization remain to be determined. Moreover, the nature and frequency of exceptions to the general patterns of grass genome structure and collinearity are still largely unknown and will require extensive further investigation.

Evolution and divergence of sodium channel genes in vertebrates

Invertebrate species possess one or two Na+ channel genes, yet there are 10 in mammals. When did this explosive growth come about during vertebrate evolution? All mammalian Na+ channel genes reside on four chromosomes. It has been suggested that this came about by multiple duplications of an ancestral chromosome with a single Na+ channel gene followed by tandem duplications of Na+ channel genes on some of these chromosomes. Because a large-scale expansion of the vertebrate genome likely occurred before the divergence of teleosts and tetrapods, we tested this hypothesis by cloning Na+ channel genes in a teleost fish. Using an approach designed to clone all of the Na+ channel genes in a genome, we found six Na+ channel genes. Phylogenetic comparisons show that each teleost gene is orthologous to a Na+ channel gene or gene cluster on a different mammalian chromosome, supporting the hypothesis that four Na+ channel genes were present in the ancestors of teleosts and tetrapods. Further duplications occurred independently in the teleost and tetrapod lineages, with a greater number of duplications in tetrapods. This pattern has implications for the evolution of function and specialization of Na+ channel genes in vertebrates. Sodium channel genes also are linked to homeobox (Hox) gene clusters in mammals. Using our phylogeny of Na+ channel genes to independently test between two models of Hox gene evolution, we support the hypothesis that Hox gene clusters evolved as (AB) (CD) rather than {D[A(BC)]}.

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.

Organization of the Hox gene cluster in the grasshopper, Schistocerca gregaria

The conserved organization of the Hox genes throughout the animal kingdom has become one of the major paradigms of evolutionary developmental biology. We have examined the organization of the Hox genes of the grasshopper, Schistocerca gregaria. We find that the grasshopper Hox cluster is over 700 kb long, and is not split into equivalents of the Antennapedia complex and the bithorax complex of Drosophila melanogaster. SgDax and probably also Sgzen, the grasshopper homologues of fushi-tarazu (ftz) and Zerknüllt (zen), respectively, are also in the cluster, showing that the non-homeotic Antp-class genes, “accessory genes,” are an ancient feature of insect Hox clusters.

Long-range disruption of gene expression by a selectable marker cassette

Recent studies have suggested that the retention of selectable marker cassettes (like PGK–Neo, in which a hybrid gene consisting of the phosphoglycerate kinase I promoter drives the neomycin phosphotransferase gene) in targeted loci can cause unexpected phenotypes in “knockout” mice due to disruption of expression of neighboring genes within a locus. We have studied targeted mutations in two multigene clusters, the granzyme B locus and the β-like globin gene cluster. The insertion of PGK–Neo into the granzyme B gene, the most 5′ gene in the granzyme B gene cluster, severely reduced the normal expression of multiple genes within the locus, even at distances greater than 100 kb from the mutation. Similarly, the insertion of a PGK–Neo cassette into the β-globin locus control region (LCR) abrogates the expression of multiple globin genes downstream from the cassette. In contrast, a targeted mutation of the promyelocyte-specific cathepsin G gene (which lies just 3′ to the granzyme genes in the same cluster) had minimal effects on upstream granzyme gene expression. Although the mechanism of these long distance effects are unknown, the expression of PGK–Neo can be “captured” by the regulatory domain into which it is inserted. These results suggest that the PGK–Neo cassette can interact productively with locus control regions and thereby disrupt normal interactions between local and long-distance regulatory regions within a tissue-specific domain.

Gene content phylogeny of herpesviruses

Clusters of orthologous groups [COGs; Tatusov, R. L., Koonin, E. V. & Lipman, D. J. (1997) Science 278, 631–637] were identified for a set of 13 completely sequenced herpesviruses. Each COG represented a family of gene products conserved across several herpes genomes. These families were defined without using an arbitrary threshold criterion based on sequence similarity. The COG technique was modified so that variable stringency in COG construction was possible. High stringencies identify a core set of highly conserved genes. Varying COG stringency reveals differences in the degree of conservation between functional classes of genes. The COG data were used to construct whole-genome phylogenetic trees based on gene content. These trees agree well with trees based on other methods and are robust when tested by bootstrap analysis. The COG data also were used to construct a reciprocal tree that clustered genes with similar phylogenetic profiles. This clustering may give clues to genes with related functions or with related histories of acquisition and loss during herpesvirus evolution.

Identification of bdm-1, a gene involved in G protein β-subunit function and α-subunit accumulation

Targeted disruption of Gα and Gβ genes has established the requirement of an intact G protein signaling pathway for optimal execution of several important physiological processes, including pathogenesis, in the chestnut blight fungus Cryphonectria parasitica. We now report the identification of a G protein signal transduction component, beta disruption mimic factor-1, BDM-1. Disruption of the corresponding gene, bdm-1, resulted in a phenotype indistinguishable from that previously observed after disruption of the Gβ subunit gene, cpgb-1. The BDM-1 deduced amino acid sequence contained several significant clusters of identity with mammalian phosducin, including a domain corresponding to a highly conserved 11-amino acid stretch that has been implicated in binding to the Gβγ dimer and two regions of defined Gβ/phosducin contact points. Unlike the negative regulatory function proposed for mammalian phosducin, the genetic data presented in this report suggest that BDM-1 is required for or facilitates Gβ function. Moreover, disruption of either bdm-1 or cpgb-1 resulted in a significant, posttranscriptional reduction in the accumulation of CPG-1, a key Gα subunit required for a range of vital physiological processes.

Genome-wide gene expression profiles of the developing mouse hippocampus

We have analyzed the developmental molecular programs of the mouse hippocampus, a cortical structure critical for learning and memory, by means of large-scale DNA microarray techniques. Of 11,000 genes and expressed sequence tags examined, 1,926 showed dynamic changes during hippocampal development from embryonic day 16 to postnatal day 30. Gene-cluster analysis was used to group these genes into 16 distinct clusters with striking patterns that appear to correlate with major developmental hallmarks and cellular events. These include genes involved in neuronal proliferation, differentiation, and synapse formation. A complete list of the transcriptional changes has been compiled into a comprehensive gene profile database (http://BrainGenomics.Princeton.edu), which should prove valuable in advancing our understanding of the molecular and genetic programs underlying both the development and the functions of the mammalian brain.