The genomic and physical organization of Ty1-copia-like sequences as a component of large genomes in Pinus elliottii var. elliottii and other gymnosperms.

A DNA sequence, TPE1, representing the internal domain of a Ty1-copia retroelement, was isolated from genomic DNA of Pinus elliottii Engelm. var. elliottii (slash pine). Genomic Southern analysis showed that this sequence, carrying partial reverse transcriptase and integrase gene sequences, is highly amplified within the genome of slash pine and part of a dispersed element >4.8 kbp. Fluorescent in situ hybridization to metaphase chromosomes shows that the element is relatively uniformly dispersed over all 12 chromosome pairs and is highly abundant in the genome. It is largely excluded from centromeric regions and intercalary chromosomal sites representing the 18S-5.8S-25S rRNA genes. Southern hybridization with specific DNA probes for the reverse transcriptase gene shows that TPE1 represents a large subgroup of heterogeneous Ty1-copia retrotransposons in Pinus species. Because no TPE1 transcription could be detected, it is most likely an inactive element--at least in needle tissue. Further evidence for inactivity was found in recombinant reverse transcriptase and integrase sequences. The distribution of TPE1 within different gymnosperms that contain Ty1-copia group retrotransposons, as shown by a PCR assay, was investigated by Southern hybridization. The TPE1 family is highly amplified and conserved in all Pinus species analyzed, showing a similar genomic organization in the three- and five-needle pine species investigated. It is also present in spruce, bald cypress (swamp cypress), and in gingko but in fewer copies and a different genomic organization.

Comparative analysis of ribonuclease P RNA using gene sequences from natural microbial populations reveals tertiary structural elements.

PCR amplification of template DNAs extracted from mixed, naturally occurring microbial populations, using oligonucleotide primers complementary to highly conserved sequences, was used to obtain a large collection of diverse RNase P RNA-encoding genes. An alignment of these sequences was used in a comparative analysis of RNase P RNA secondary and tertiary structure. The new sequences confirm the secondary structure model based on sequences from cultivated organisms (with minor alterations in helices P12 and P18), providing additional support for nearly every base pair. Analysis of sequence covariation using the entire RNase P RNA data set reveals elements of tertiary structure in the RNA; the third nucleotides (underlined) of the GNRA tetraloops L14 and L18 are seen to interact with adjacent Watson-Crick base pairs in helix P8, forming A:G/C or G:A/U base triples. These experiments demonstrate one way in which the enormous diversity of natural microbial populations can be used to elucidate molecular structure through comparative analysis.

Q/R site editing in kainate receptor GluR5 and GluR6 pre-mRNAs requires distant intronic sequences.

RNA editing by adenosine deamination in brain-expressed pre-mRNAs for glutamate receptor (GluR) subunits alters gene-specified codons for functionally critical positions, such as the channel's Q/R site. We show by transcript analysis of minigenes transiently expressed in PC-12 cells that, in contrast to GluR-B pre-mRNA, where the two editing sites (Q/R and R/G) require base pairing with nearby intronic editing site complementary sequences (ECSs), editing in GluR5 and GluR6 pre-mRNAs recruits an ECS located as far as 1900 nucleotides distal to the Q/R site. The exon-intron duplex structure of the GluR5 and GluR6 pre-mRNAs appears to be a substrate of double-stranded RNA-specific adenosine deaminase. This enzyme when coexpressed in HEK 293 cells preferentially targets the adenosine of the Q/R site and of an unpaired position in the ECS which is highly edited in brain.

Recognition of diverse sequences by class I zinc fingers: asymmetries and indirect effects on specificity in the interaction between CF2II and A+T-rich elements.

The Drosophila CF2II protein, which contains zinc fingers of the Cys2His2 type and recognizes an A+T-rich sequence, behaves in cell culture as an activator of a reporter chloramphenicol acetyltransferase gene. This activity depends on C-terminal but not N-terminal zinc fingers, as does in vitro DNA binding. By site-specific mutagenesis and binding site selection, we define the critical amino acid-base interactions. Mutations of single amino acid residues at the leading edge of the recognition helix are rarely neutral: many result in a slight change in affinity for the ideal DNA target site; some cause major loss of affinity; and others change specificity for as many as two bases in the target site. Compared to zinc fingers that recognize G+C-rich DNA, CF2II fingers appear to bind to A+T-rich DNA in a generally similar manner, but with additional flexibility and amino acid-base interactions. The results illustrate how zinc fingers may be evolving to recognize an unusually diverse set of DNA sequences.

Adaptive mutation sequences reproduced by mismatch repair deficiency.

Adaptive reversions of a lac frameshift mutation in Escherichia coli are -1 deletions in small mononucleotide repeats, whereas growth-dependent reversions are heterogeneous. The adaptive mutations resemble instability of simple repeats, which, in hereditary colon cancer, in yeast, and in E. coli occurs in the absence of mismatch repair. The postulate that mismatch repair is disabled transiently during adaptive mutation in E. coli is supported here by the demonstration that the growth-dependent mutation spectrum can be made indistinguishable from adaptive mutations by disallowing mismatch repair during growth. Physiologically induced mismatch repair deficiency could be an important mutagenic mechanism in cancers and in evolution.

Contrasting histories of avian and mammalian Mhc genes revealed by class II B sequences from songbirds.

To explore the evolutionary dynamics of genes in the major histocompatibility complex (Mhc) in nonmammalian vertebrates, we have amplified complete sequences of the polymorphic second (beta1) and third (beta2) exons of class II beta chain genes of songbirds. The pattern of nucleotide substitution in the antigen-binding site of sequences cloned from three behaviorally and phylogenetically divergent songbirds [scrub jays Aphelocoma coerulescens), red-winged blackbirds (Agelaius phoeniceus), and house finches (Carpodacus mexicanus) reveals that class II B genes of songbirds are subject to the same types of diversifying forces as those observed at mammalian class II loci. By contrast, the tree of avian class II B genes reveals that orthologous relationships have not been retained as in placental mammals and that, unlike class II genes in mammals, genes in songbirds and chickens have had very recent common ancestors within their respective groups. Thus, whereas the selective forces diversifying class II B genes of birds are likely similar to those in mammals, their long-term evolutionary dynamics appear to be characterized by much higher rates of concerted evolution.

Isolation of virus-neutralizing RNAs from a large pool of random sequences.

RNA and ribonuclease-resistant RNA analogs that bound and neutralized Rous sarcoma virus (RSV) were isolated from a large pool of random sequences by multiple cycles of in vitro selection using infectious viral particles. The selected RNA pool of RSV-binding sequences at a concentration of 0.16 microM completely neutralized the virus. Of 19 sequences cloned from the selected pool, 5 inhibited RSV infection. The selected RNA and RNA analogs were shown to neutralize RSV by interacting with the virus, rather than by adversely affecting the host cells. The selection of the anti-RSV RNA and RNA analogs by intact virions immediately suggests the potential application of this approach to develop RNA and RNA analogs as inhibitors of other viruses such as human immunodeficiency virus.

Restriction-modification systems as genomic parasites in competition for specific sequences.

Restriction-modification (RM) systems are believed to have evolved to protect cells from foreign DNA. However, this hypothesis may not be sufficient to explain the diversity and specificity in sequence recognition, as well as other properties, of these systems. We report that the EcoRI restriction endonuclease-modification methylase (rm) gene pair stabilizes plasmids that carry it and that this stabilization is blocked by an RM of the same sequence specificity (EcoRI or its isoschizomer, Rsr I) but not by an RM of a different specificity (PaeR7I) on another plasmid. The PaeR7I rm likewise stabilizes plasmids, unless an rm gene pair with identical sequence specificity is present. Our analysis supports the following model for stabilization and incompatibility: the descendants of cells that have lost an rm gene pair expose the recognition sites in their chromosomes to lethal attack by any remaining restriction enzymes unless modification by another RM system of the same specificity protects these sites. Competition for specific sequences among these selfish genes may have generated the great diversity and specificity in sequence recognition among RM systems. Such altruistic suicide strategies, similar to those found in virus-infected cells, may have allowed selfish RM systems to spread by effectively competing with other selfish genes.

Inferring phylogenies from DNA sequences of unequal base compositions.

A new method for computing evolutionary distances between DNA sequences is proposed. Contrasting with classical methods, the underlying model does not assume that sequence base compositions (A, C, G, and T contents) are at equilibrium, thus allowing unequal base compositions among compared sequences. This makes the method more efficient than the usual ones in recovering phylogenetic trees from sequence data when base composition is heterogeneous within the data set, as we show by using both simulated and empirical data. When applied to small-subunit ribosomal RNA sequences from several prokaryotic or eukaryotic organisms, this method provides evidence for an early divergence of the microsporidian Vairimorpha necatrix in the eukaryotic lineage.

Signaling by N- and C-terminal sequences of parathyroid hormone-related protein in hippocampal neurons.

Parathyroid hormone-related protein (PTHrP) is synthesized in the brain, and a single type of cloned receptor for the N-terminal portion of PTHrP and PTH is present in the central nervous system. Nothing is known about the physiological actions or signaling pathways used by PTHrP in the brain. Using cultured rat hippocampal neurons, we demonstrate that N-terminal PTHrP[1-34] and PTH[1-34] signal via cAMP and cytosolic calcium transients. The cAMP response showed strong acute (< or = 6 h) homologous and heterologous desensitization after preincubation with PTHrP or PTH. In contrast, the acute calcium response did not desensitize after preincubation with PTHrP; in fact, preincubation dramatically recruited additional responsive neurons. Unexpectedly, C-terminal PTHrP[107-139], which does not bind or activate the cloned PTH/PTHrP receptor, signaled in neurons via cytosolic calcium but not cAMP. Although some neurons responded to both PTHrP[1-34] and PTHrP[107-139], others responded only to PTHrP[1-34]. We conclude that certain hippocampal neurons exhibit dual signaling in response to PTHrP[1-34] and that some neurons have a receptor for C-terminal PTHrP that signals only via cytosolic calcium.

[Leu5]enkephalin-encoding sequences are targets for a specific DNA-binding factor.

A DNA-binding factor with high affinity and specificity for the [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes has been characterized. The factor has the highest affinity for the [Leu5]-enkephalin-encoding sequence in the dynorphin B-encoding region of the prodynorphin gene, has relatively high affinity for other [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes, but has no apparent affinity for similar DNA sequences coding for [Met5]-enkephalin in the prodynorphin or proopiomelanocortin genes. The factor has been named [Leu5]enkephalin-encoding sequence DNA-binding factor (LEF). LEF has a nuclear localization and is composed of three subunits of about 60, 70, and 95 kDa, respectively. The highest levels were observed in rat testis, cerebellum, and spleen and were generally higher in late embryonal compared to newborn or adult animals. LEF activity was also recorded in human clonal tumor cell lines. LEF inhibited the transcription of reporter genes in artificial gene constructs where a [Leu5]enkephalin-encoding DNA fragment had been inserted between the transcription initiation site and the coding region of the reporter genes. These observations suggest that the [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes also have regulatory functions realized through interaction with a specific DNA-binding factor.

Regulation of glycolipid synthesis in HL-60 cells by antisense oligodeoxynucleotides to glycosyltransferase sequences: effect on cellular differentiation.

Treatment of the human promyelocytic leukemia cell line HL-60 with antisense oligodeoxynucleotides to UDP-N-acetylgalactosamine:beta-1,4-N-acetylgalactosaminyl-transferase (GM2-synthase; EC 2.4.1.92) and CMP-sialic acid:alpha-2,8-sialyltransferase (GD3-synthase; EC 2.4.99.8) sequences effectively down-regulated the synthesis of more complex gangliosides in the ganglioside synthetic pathways after GM3, resulting in a remarkable increase in endogenous GM3 with concomitant decreases in more complex gangliosides. The treated cells underwent monocytic differentiation as judged by morphological changes, adherent ability, and nitroblue tetrazolium staining. These data provide evidence that the increased endogenous ganglioside GM3 may play an important role in regulating cellular differentiation and that the antisense DNA technique proves to be a powerful tool in manipulating glycolipid synthesis in the cell.

Toward the therapeutic editing of mutated RNA sequences.

If RNA editing could be rationally directed to mutated RNA sequences, genetic diseases caused by certain base substitutions could be treated. Here we use a synthetic complementary RNA oligonucleotide to direct the correction of a premature stop codon mutation in dystrophin RNA. The complementary RNA oligonucleotide was hybridized to a premature stop codon and the hybrid was treated with nuclear extracts containing the cellular enzyme double-stranded RNA adenosine deaminase. When the treated RNAs were translated in vitro, a dramatic increase in expression of a downstream luciferase coding region was observed. The cDNA sequence data are consistent with deamination of the adenosine in the UAG stop codon to inosine by double-stranded RNA adenosine deaminase. Injection of oligonucleotide-mRNA hybrids into Xenopus embryos also resulted in an increase in luciferase expression. These experiments demonstrate the principle of therapeutic RNA editing.

Conformation, energy, and folding ability of selected amino acid sequences.

Evolutionary selection of sequences is studied with a knowledge-based Hamiltonian to find the design principle for folding to a model protein structure. With sequences selected by naive energy minimization, the model structure tends to be unstable and the folding ability is low. Sequences with high folding ability have only the low-lying energy minimum but also an energy landscape which is similar to that found for the native sequence over a wide region of the conformation space. Though there is a large fluctuation in foldable sequences, the hydrophobicity pattern and the glycine locations are preserved among them. Implications of the design principle for the molecular mechanism of folding are discussed.

Identification of planarian homeobox sequences indicates the antiquity of most Hox/homeotic gene subclasses.

The homeotic gene complex (HOM-C) is a cluster of genes involved in the anteroposterior axial patterning of animal embryos. It is composed of homeobox genes belonging to the Hox/HOM superclass. Originally discovered in Drosophila, Hox/HOM genes have been identified in organisms as distantly related as arthropods, vertebrates, nematodes, and cnidarians. Data obtained in parallel from the organization of the complex, the domains of gene expression during embryogenesis, and phylogenetic relationships allow the subdivision of the Hox/HOM superclass into five classes (lab, pb/Hox3, Dfd, Antp, and Abd-B) that appeared early during metazoan evolution. We describe a search for homologues of these genes in platyhelminths, triploblast metazoans emerging as an outgroup to the great coelomate ensemble. A degenerate PCR screening for Hox/HOM homeoboxes in three species of triclad planarians has revealed 10 types of Antennapedia-like genes. The homeobox-containing sequences of these PCR fragments allowed the amplification of the homeobox-coding exons for five of these genes in the species Polycelis nigra. A phylogenetic analysis shows that two genes are clear orthologues of Drosophila labial, four others are members of a Dfd/Antp superclass, and a seventh gene, although more difficult to classify with certainty, may be related to the pb/Hox3 class. Together with previously identified Hox/HOM genes in other flatworms, our analyses demonstrate the existence of an elaborate family of Hox/HOM genes in the ancestor of all triploblast animals.