Structural basis of an embryonically lethal single Ala → Thr mutation in the vnd/NK-2 homeodomain

The structural and DNA binding behavior is described for an analog of the vnd/NK-2 homeodomain, which contains a single amino acid residue alanine to threonine replacement in position 35 of the homeodomain. Multidimensional nuclear magnetic resonance, circular dichroism, and electrophoretic gel retardation assays were carried out on recombinant 80-aa residue proteins that encompass the wild-type and mutant homeodomains. The mutant A35T vnd/NK-2 homeodomain is unable to adopt a folded conformation free in solution at temperatures down to −5°C in contrast to the behavior of the corresponding wild-type vnd/NK-2 homeodomain, which is folded into a functional three-dimensional structure below 25°C. The A35T vnd/NK-2 binds specifically to the vnd/NK-2 target DNA sequence, but with an affinity that is 50-fold lower than that of the wild-type homeodomain. Although the three-dimensional structure of the mutant A35T vnd/NK-2 in the DNA bound state shows characteristic helix–turn–helix behavior similar to that of the wild-type homeodomain, a notable structural deviation in the mutant A35T analog is observed for the amide proton of leucine-40. The wild-type homeodomain forms an unusual i,i-5 hydrogen bond with the backbone amide oxygen of residue 35. In the A35T mutant this amide proton resonance is shifted upfield by 1.27 ppm relative to the resonance frequency for the wild-type analog, thereby indicating a significant alteration of this i,i-5 hydrogen bond.

SIRE-1, a copia/Ty1-like retroelement from soybean, encodes a retroviral envelope-like protein

The soybean genome hosts a family of several hundred, relatively homogeneous copies of a large, copia/Ty1-like retroelement designated SIRE-1. A copy of this element has been recovered from a Glycine max genomic library. DNA sequence analysis of two SIRE-1 subclones revealed that SIRE-1 contains a long, uninterrupted, ORF between the 3′ end of the pol ORF and the 3′ long terminal repeat (LTR), a region that harbors the env gene in retroviral genomes. Conceptual translation of this second ORF produces a 70-kDa protein. Computer analyses of the amino acid sequence predicted patterns of transmembrane domains, α-helices, and coiled coils strikingly similar to those found in mammalian retroviral envelope proteins. In addition, a 65-residue, proline-rich domain is characterized by a strong amino acid compositional bias virtually identical to that of the 60-amino acid, proline-rich neutralization domain of the feline leukemia virus surface protein. The assignment of SIRE-1 to the copia/Ty1 family was confirmed by comparison of the conceptual translation of its reverse transcriptase-like domain with those of other retroelements. This finding suggests the presence of a proretrovirus in a plant genome and is the strongest evidence to date for the existence of a retrovirus-like genome closely related to copia/Ty1 retrotransposons.

Discrimination of the closely related A and D genomes of the hexaploid oat Avena sativa L.

A satellite DNA sequence, As120a, specific to the A-genome chromosomes in the hexaploid oat, Avena sativa L., was isolated by subcloning a fragment with internal tandem repeats from a plasmid, pAs120, that had been obtained from an Avena strigosa (As genome) genomic library. Southern and in situ hybridization showed that sequences with homology to sequences within pAs120 were dispersed throughout the genome of diploid (A and C genomes), tetraploid (AC genomes), and hexaploid (ACD genomes) Avena species. In contrast, sequences homologous to As120a were found in two A-genome species (A. strigosa and Avena longiglumis) and in the hexaploid A. sativa whereas this sequence was little amplified in the tetraploid Avena murphyi and was absent in the remaining A- and C-genome diploid species. In situ hybridization of pAs120a to hexaploid oat species revealed the distribution of elements of the As120a repeated family over both arms of 14 of 42 chromosomes of this species. By using double in situ hybridization with pAs120a and a C genome-specific probe, three sets of 14 chromosomes were revealed corresponding to the A, C, and D genomes of the hexaploid species. Simultaneous in situ hybridizations with pAs120a and ribosomal probes were used to assign the SAT chromosomes of hexaploid species to their correct genomes. This work reports a sequence able to distinguish between the closely related A and D genomes of hexaploid oats. This sequence offers new opportunities to analyze the relationships of Avena species and to explore the possible evolution of various polyploid oat species.

Structural flexibility in transcription complex formation revealed by protein–DNA photocrosslinking

The Oct-1 POU domain binds diverse DNA-sequence elements and forms a higher-order regulatory complex with the herpes simplex virus coregulator VP16. The POU domain contains two separate DNA-binding domains joined by a flexible linker. By protein–DNA photocrosslinking we show that the relative positioning of the two POU DNA-binding domains on DNA varies depending on the nature of the DNA target. On a single VP16-responsive element, the POU domain adopts multiple conformations. To determine the structure of the Oct-1 POU domain in a multiprotein complex with VP16, we allowed VP16 to interact with previously crosslinked POU-domain–DNA complexes and found that VP16 can associate with multiple POU-domain conformations. These results reveal the dynamic potential of a DNA-binding domain in directing transcriptional regulatory complex formation.

Two living species of coelacanths?

During the period of September 1997 through July 1998, two coelacanth fishes were captured off Manado Tua Island, Sulawesi, Indonesia. These specimens were caught almost 10,000 km from the only other known population of living coelacanths, Latimeria chalumnae, near the Comores. The Indonesian fish was described recently as a new species, Latimeria menadoensis, based on morphological differentiation and DNA sequence divergence in fragments of the cytochrome b and 12S rRNA genes. We have obtained the sequence of 4,823 bp of mitochondrial DNA from the same specimen, including the entire genes for cytochrome b, 12S rRNA, 16S rRNA, four tRNAs, and the control region. The sequence is 4.1% different from the published sequence of an animal captured from the Comores, indicating substantial divergence between the Indonesian and Comorean populations. Nine morphological and meristic differences are purported to distinguish L. menadoensis and L. chalumnae, based on comparison of a single specimen of L. menadoensis to a description of five individuals of L. chalumnae from the Comores. A survey of the literature provided data on 4 of the characters used to distinguish L. menadoensis from L. chalumnae from an additional 16 African coelacanths; for all 4 characters, the Indonesian sample was within the range of variation reported for the African specimens. Nonetheless, L. chalumnae and L. menadoensis appear to be separate species based on divergence of mitochondrial DNA.

Barriers to recombination between closely related bacteria: MutS and RecBCD inhibit recombination between Salmonella typhimurium and Salmonella typhi

Previous studies have shown that inactivation of the MutS or MutL mismatch repair enzymes increases the efficiency of homeologous recombination between Escherichia coli and Salmonella typhimurium and between S. typhimurium and Salmonella typhi. However, even in mutants defective for mismatch repair the recombination frequencies are 102- to 103-fold less than observed during homologous recombination between a donor and recipient of the same species. In addition, the length of DNA exchanged during transduction between S. typhimurium and S. typhi is less than in transductions between strains of S. typhimurium. In homeologous transductions, mutations in the recD gene increased the frequency of transduction and the length of DNA exchanged. Furthermore, in mutS recD double mutants the frequency of homeologous recombination was nearly as high as that seen during homologous recombination. The phenotypes of the mutants indicate that the gene products of mutS and recD act independently. Because S. typhimurium and S. typhi are ≈98–99% identical at the DNA sequence level, the inhibition of recombination is probably not due to a failure of RecA to initiate strand exchange. Instead, these results suggest that mismatches act at a subsequent step, possibly by slowing the rate of branch migration. Slowing the rate of branch migration may stimulate helicase proteins to unwind rather than extend the heteroduplex and leave uncomplexed donor DNA susceptible to further degradation by RecBCD exonuclease.

Compositional differences within and between eukaryotic genomes

Eukaryotic genome similarity relationships are inferred using sequence information derived from large aggregates of genomic sequences. Comparisons within and between species sample sequences are based on the profile of dinucleotide relative abundance values (The profile is ρ*XY = f*XY/f*Xf*Y for all XY, where f*X denotes the frequency of the nucleotide X and f*XY denotes the frequency of the dinucleotide XY, both computed from the sequence concatenated with its inverted complement). Previous studies with respect to prokaryotes and this study document that profiles of different DNA sequence samples (sample size ≥50 kb) from the same organism are generally much more similar to each other than they are to profiles from other organisms, and that closely related organisms generally have more similar profiles than do distantly related organisms. On this basis we refer to the collection {ρ*XY} as the genome signature. This paper identifies ρ*XY extremes and compares genome signature differences for a diverse range of eukaryotic species. Interpretations on the mechanisms maintaining these profile differences center on genome-wide replication, repair, DNA structures, and context-dependent mutational biases. It is also observed that mitochondrial genome signature differences between species parallel the corresponding nuclear genome signature differences despite large differences between corresponding mitochondrial and nuclear signatures. The genome signature differences also have implications for contrasts between rodents and other mammals, and between monocot and dicot plants, as well as providing evidence for similarities among fungi and the diversity of protists.

Convergent evolution of apolipoprotein(a) in primates and hedgehog

Apolipoprotein(a) [apo(a)] is the distinguishing protein component of lipoprotein(a), a major inherited risk factor for atherosclerosis. Human apo(a) is homologous to plasminogen. It contains from 15 to 50 repeated domains closely related to plasminogen kringle four, plus single kringle five-like and inactive protease-like domains. This expressed gene is confined to a subset of primates. Although most mammals lack apo(a), hedgehogs produce an apo(a)-like protein composed of highly repeated copies of a plasminogen kringle three-like domain, with complete absence of protease domain sequences. Both human and hedgehog apo(a)-like proteins form covalently linked lipoprotein particles that can bind to fibrin and other substrates shared with plasminogen. DNA sequence comparisons and phylogenetic analysis indicate that the human type of apo(a) evolved from a duplicated plasminogen gene during recent primate evolution. In contrast, the kringle three-based type of apo(a) evolved from an independent duplication of the plasminogen gene approximately 80 million years ago. In a type of convergent evolution, the plasminogen gene has been independently remodeled twice during mammalian evolution to produce similar forms of apo(a) in two widely divergent groups of species.

Chromosome-specific molecular organization of maize (Zea mays L.) centromeric regions

A set of oat–maize chromosome addition lines with individual maize (Zea mays L.) chromosomes present in plants with a complete oat (Avena sativa L.) chromosome complement provides a unique opportunity to analyze the organization of centromeric regions of each maize chromosome. A DNA sequence, MCS1a, described previously as a maize centromere-associated sequence, was used as a probe to isolate cosmid clones from a genomic library made of DNA purified from a maize chromosome 9 addition line. Analysis of six cosmid clones containing centromeric DNA segments revealed a complex organization. The MCS1a sequence was found to comprise a portion of the long terminal repeats of a retrotransposon-like repeated element, termed CentA. Two of the six cosmid clones contained regions composed of a newly identified family of tandem repeats, termed CentC. Copies of CentA and tandem arrays of CentC are interspersed with other repetitive elements, including the previously identified maize retroelements Huck and Prem2. Fluorescence in situ hybridization revealed that CentC and CentA elements are limited to the centromeric region of each maize chromosome. The retroelements Huck and Prem2 are dispersed along all maize chromosomes, although Huck elements are present in an increased concentration around centromeric regions. Significant variation in the size of the blocks of CentC and in the copy number of CentA elements, as well as restriction fragment length variations were detected within the centromeric region of each maize chromosome studied. The different proportions and arrangements of these elements and likely others provide each centromeric region with a unique overall structure.

Gene-based approach to human gene-phenotype correlations

Elucidating the genetic basis of human phenotypes is a major goal of contemporary geneticists. Logically, two fundamental and contrasting approaches are available, one that begins with a phenotype and concludes with the identification of a responsible gene or genes; the other that begins with a gene and works toward identifying one or more phenotypes resulting from allelic variation of it. This paper provides a conceptual overview of phenotype-based vs. gene-based procedures with emphasis on gene-based methods. A key feature of a gene-based approach is that laboratory effort first is devoted to developing an assay for mutations in the gene under regard; the assay then is applied to the evaluation of large numbers of unrelated individuals with a variety of phenotypes that are deemed potentially resulting from alleles at the gene. No effort is directed toward chromosomally mapping the loci responsible for the phenotypes scanned. Example is made of my laboratory’s successful use of a gene-based approach to identify genes causing hereditary diseases of the retina such as retinitis pigmentosa. Reductions in the cost and improvements in the speed of scanning individuals for DNA sequence anomalies may make a gene-based approach an efficient alternative to phenotype-based approaches to correlating genes with phenotypes.

PCR-based subtractive hybridization and differences in gene content among strains of Helicobacter pylori

Genes that are characteristic of only certain strains of a bacterial species can be of great biologic interest. Here we describe a PCR-based subtractive hybridization method for efficiently detecting such DNAs and apply it to the gastric pathogen Helicobacter pylori. Eighteen DNAs specific to a monkey-colonizing strain (J166) were obtained by subtractive hybridization against an unrelated strain whose genome has been fully sequenced (26695). Seven J166-specific clones had no DNA sequence match to the 26695 genome, and 11 other clones were mixed, with adjacent patches that did and did not match any sequences in 26695. At the protein level, seven clones had homology to putative DNA restriction-modification enzymes, and two had homology to putative metabolic enzymes. Nine others had no database match with proteins of assigned function. PCR tests of 13 unrelated H. pylori strains by using primers specific for 12 subtracted clones and complementary Southern blot hybridizations indicated that these DNAs are highly polymorphic in the H. pylori population, with each strain yielding a different pattern of gene-specific PCR amplification. The search for polymorphic DNAs, as described here, should help identify previously unknown virulence genes in pathogens and provide new insights into microbial genetic diversity and evolution.

ETS target genes: Identification of Egr1 as a target by RNA differential display and whole genome PCR techniques

ETS transcription factors play important roles in hematopoiesis, angiogenesis, and organogenesis during murine development. The ETS genes also have a role in neoplasia, for example in Ewing’s sarcomas and retrovirally induced cancers. The ETS genes encode transcription factors that bind to specific DNA sequences and activate transcription of various cellular and viral genes. To isolate novel ETS target genes, we used two approaches. In the first approach, we isolated genes by the RNA differential display technique. Previously, we have shown that the overexpression of ETS1 and ETS2 genes effects transformation of NIH 3T3 cells and specific transformants produce high levels of the ETS proteins. To isolate ETS1 and ETS2 responsive genes in these transformed cells, we prepared RNA from ETS1, ETS2 transformants, and normal NIH 3T3 cell lines and converted it into cDNA. This cDNA was amplified by PCR and displayed on sequencing gels. The differentially displayed bands were subcloned into plasmid vectors. By Northern blot analysis, several clones showed differential patterns of mRNA expression in the NIH 3T3-, ETS1-, and ETS2-expressing cell lines. Sixteen clones were analyzed by DNA sequence analysis, and 13 of them appeared to be unique because their DNA sequences did not match with any of the known genes present in the gene bank. Three known genes were found to be identical to the CArG box binding factor, phospholipase A2-activating protein, and early growth response 1 (Egr1) genes. In the second approach, to isolate ETS target promoters directly, we performed ETS1 binding with MboI-cleaved genomic DNA in the presence of a specific mAb followed by whole genome PCR. The immune complex-bound ETS binding sites containing DNA fragments were amplified and subcloned into pBluescript and subjected to DNA sequence and computer analysis. We found that, of a large number of clones isolated, 43 represented unique sequences not previously identified. Three clones turned out to contain regulatory sequences derived from human serglycin, preproapolipoprotein C II, and Egr1 genes. The ETS binding sites derived from these three regulatory sequences showed specific binding with recombinant ETS proteins. Of interest, Egr1 was identified by both of these techniques, suggesting strongly that it is indeed an ETS target gene.

Photorepair mutants of Arabidopsis

UV radiation induces two major DNA damage products, the cyclobutane pyrimidine dimer (CPD) and, at a lower frequency, the pyrimidine (6–4) pyrimidinone dimer (6–4 product). Although Escherichia coli and Saccharomyces cerevisiae produce a CPD-specific photolyase that eliminates only this class of dimer, Arabidopsis thaliana, Drosophila melanogaster, Crotalus atrox, and Xenopus laevis have recently been shown to photoreactivate both CPDs and 6–4 products. We describe the isolation and characterization of two new classes of mutants of Arabidopsis, termed uvr2 and uvr3, that are defective in the photoreactivation of CPDs and 6–4 products, respectively. We demonstrate that the CPD photolyase mutation is genetically linked to a DNA sequence encoding a type II (metazoan) CPD photolyase. In addition, we are able to generate plants in which only CPDs or 6–4 products are photoreactivated in the nuclear genome by exposing these mutants to UV light and then allowing them to repair one or the other class of dimers. This provides us with a unique opportunity to study the biological consequences of each of these two major UV-induced photoproducts in an intact living system.

Up-regulation of specific tyrosinase mRNAs in mouse melanomas with the c2j gene substituted for the wild-type tyrosinase allele: Utilization in design of syngeneic immunotherapy models

The expression of cell-specialization genes is likely to be changing in tumor cells as their differentiation declines. Functional changes in these genes might yield unusual peptide epitopes with anti-tumor potential and could occur without modification in the DNA sequence of the gene. Melanomas undergo a characteristic decline in melanization that may reflect altered contributions of key melanocytic genes such as tyrosinase. Quantitative reverse transcriptase–PCR of the wild-type (C) tyrosinase gene in transgenic (C57BL/6 strain) mouse melanomas has revealed a shift toward alternative splicing of the pre-mRNA that generated increased levels of the Δ1b and Δ1d mRNA splice variants. The spontaneous c2j albino mutation of tyrosinase (in the C57BL/6 strain) changes the pre-mRNA splicing pattern. In c2j/c2j melanomas, alternative splicing was again increased. However, while some mRNAs (notably Δ1b) present in C/C were obligatorily absent, others (Δ3 and Δ1d) were elevated. In c2j/c2j melanomas, the percentage of total tyrosinase transcripts attributable to Δ3 reached approximately 2-fold the incidence in c2j/c2j or C/C skin melanocytes. The percentage attributable to Δ1d rose to approximately 2-fold the incidence in c2j/c2j skin, and to 10-fold that in C/C skin. These differences provide a basis for unique mouse models in which the melanoma arises in skin grafted from a C/C or c2j/c2j transgenic donor to a transgenic host of the same or opposite tyrosinase genotype. Immunotherapy designs then could be based on augmenting those antigenic peptides that are novel or overrepresented in a tumor relative to the syngeneic host.

A test of alternative models of diversification in tropical rainforests: Ecological gradients vs. rainforest refugia

Comparison of mitochondrial and morphological divergence in eight populations of a widespread leaf-litter skink is used to determine the relative importance of geographic isolation and natural selection in generating phenotypic diversity in the Wet Tropics Rainforest region of Australia. The populations occur in two geographically isolated regions, and within each region, in two different habitats (closed rainforest and tall open forest) that span a well characterized ecological gradient. Morphological differences among ancient geographic isolates (separated for several million years, judging by their mitochondrial DNA sequence divergence) were slight, but morphological and life history differences among habitats were large and occurred despite moderate to high levels of mitochondrial gene flow. A field experiment identified avian predation as one potential agent of natural selection. These results indicate that natural selection operating across ecological gradients can be more important than geographic isolation in similar habitats in generating phenotypic diversity. In addition, our results indicate that selection is sufficiently strong to overcome the homogenizing effects of gene flow, a necessary first step toward speciation in continuously distributed populations. Because ecological gradients may be a source of evolutionary novelty, and perhaps new species, their conservation warrants greater attention. This is particularly true in tropical regions, where most reserves do not include ecological gradients and transitional habitats.