Flightless brown kiwis of New Zealand possess extremely subdivided population structure and cryptic species like small mammals.

Using allozymes and mtDNA sequences from the cytochrome b gene, we report that the brown kiwi has the highest levels of genetic structuring observed in birds. Moreover, the mtDNA sequences are, with two minor exceptions, diagnostic genetic markers for each population investigated, even though they are among the more slowly evolving coding regions in this genome. A major unexpected finding was the concordant split in molecular phylogenies between brown kiwis in the southern South Island and elsewhere in New Zealand. This basic phylogeographic boundary halfway down the South Island coincides with a fixed allele difference in the Hb nuclear locus and strongly suggests that two morphologically cryptic species are currently merged under one polytypic species. This is another striking example of how molecular genetic assays can detect phylogenetic discontinuities that are not reflected in traditional morphologically based taxonomies. However, reanalysis of the morphological characters by using phylogenetic methods revealed that the reason for this discordance is that most are primitive and thus are phylogenetically uninformative. Shared-derived morphological characters support the same relationships evident in the molecular phylogenies and, in concert with the molecular data, suggest that as brown kiwis colonized northward from the southern South Island, they retained many primitive characters that confounded earlier systematists. Strong subdivided population structure and cryptic species in brown kiwis seem to have evolved relatively recently as a consequence of Pleistocene range disjunctions, low dispersal power, and genetic drift in small populations.

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.

Sequence analysis of the cis-regulatory regions of the bithorax complex of Drosophila.

The bithorax complex (BX-C) of Drosophila, one of two complexes that act as master regulators of the body plan of the fly, has now been entirely sequenced and comprises approximately 315,000 bp, only 1.4% of which codes for protein. Analysis of this sequence reveals significantly overrepresented DNA motifs of unknown, as well as known, functions in the non-protein-coding portion of the sequence. The following types of motifs in that portion are analyzed: (i) concatamers of mono-, di-, and trinucleotides; (ii) tightly clustered hexanucleotides (spaced < or = 5 bases apart); (iii) direct and reverse repeats longer than 20 bp; and (iv) a number of motifs known from biochemical studies to play a role in the regulation of the BX-C. The hexanucleotide AGATAC is remarkably overrepresented and is surmised to play a role in chromosome pairing. The positions of sites of highly overrepresented motifs are plotted for those that occur at more than five sites in the sequence, when < 0.5 case is expected. Expected values are based on a third-order Markov chain, which is the optimal order for representing the BXCALL sequence.

Cloning of an organic solvent-resistance gene in Escherichia coli: the unexpected role of alkylhydroperoxide reductase.

Although bacterial strain able to grow in the presence of organic solvents have been isolated, little is known about the mechanism of their resistance. In the present study, 1,2,3,4-tetrahydronaphthalene (tetralin), a solvent with potential applications in industrial biocatalysis, was used to select a resistant mutant of Escherichia coli. The resultant mutant strain was tested for resistance to a wide range of solvents of varying hydrophobicities and was found to be resistant not only to tetralin itself but also to cyclohexane, propylbenzene, and 1,2-dihydronaphthalene. A recombinant library from mutant DNA was used to clone the resistance gene. The sequence of the cloned locus was determined and found to match the sequence of the previously described alkylhydroperoxide reductase operon ahpCF. The mutation was localized to a substitution of valine for glycine at position 142 in the coding region of ahpC, which is the gene encoding the catalytic subunit of the enzyme. The ahpC mutant was found to have an activity that was three times that of the wild type in reducing tetralin hydroperoxide to 1,2,3,4-tetrahydro-1-naphthol. We conclude that the toxicity of such solvents as tetralin is caused by the formation of toxic hydroperoxides in the cell. The ahpC mutation increases the activity of the enzyme toward hydrophobic hydroperoxides, thereby conferring resistance. The ahpC mutant was sensitive to the more hydrophilic solvents xylene and toluene, suggesting that there are additional mechanisms of solvent toxicity. Mutants resistant to a mixture of xylene and tetralin were isolated from the ahpC mutant but not from the wild-type strain.

Phenotype correction in retinal pigment epithelium in murine mucopolysaccharidosis VII by adenovirus-mediated gene transfer.

We have studied the use of adenovirus-mediated gene transfer to reverse the pathologic changes of lysosomal storage disease caused by beta-glucuronidase deficiency in the eyes of mice with mucopolysaccharidosis VII. A recombinant adenovirus carrying the human beta-glucuronidase cDNA coding region under the control of a non-tissue-specific promoter was injected intravitreally or subretinally into the eyes of mice with mucopolysaccharidosis VII. At 1-3 weeks after injection, the treated and control eyes were examined histochemically for beta-glucuronidase expression and histologically for phenotypic correction of the lysosomal storage defect. Enzymatic expression was detected 1-3 weeks after injection. Storage vacuoles in the retinal pigment epithelium (RPE) were still present 1 week after gene transfer but were reduced to undetectable levels by 3 weeks in both intravitreally and subretinally injected eyes. There was minimal evidence of ocular pathology associated with the viral injection. These data indicate that adenovirus-mediated gene transfer to the eye may provide for adjunctive therapy for lysosomal storage diseases affecting the RPE in conjunction with enzyme replacement and/or gene therapies for correction of systemic disease manifestations. The data also support the view that recombinant adenovirus may be useful as a gene therapy vector for retinal degenerations that result from a primary genetic defect in the RPE cells.

Structural organization of mouse peroxisome proliferator-activated receptor gamma (mPPAR gamma) gene: alternative promoter use and different splicing yield two mPPAR gamma isoforms.

To gain insight into the regulation of expression of peroxisome proliferator-activated receptor (PPAR) isoforms, we have determined the structural organization of the mouse PPAR gamma (mPPAR gamma) gene. This gene extends > 105 kb and gives rise to two mRNAs (mPPAR gamma 1 and mPPAR gamma 2) that differ at their 5' ends. The mPPAR gamma 2 cDNA encodes an additional 30 amino acids N-terminal to the first ATG codon of mPPAR gamma 1 and reveals a different 5' untranslated sequence. We show that mPPAR gamma 1 mRNA is encoded by eight exons, whereas the mPPAR gamma 2 mRNA is encoded by seven exons. Most of the 5' untranslated sequence of mPPAR gamma 1 mRNA is encoded by two exons, whereas the 5' untranslated sequence and the extra 30 N-terminal amino acids of mPPAR gamma 2 are encoded by one exon, which is located between the second and third exons coding for mPPAR gamma 1. The last six exons of mPPAR gamma gene code for identical sequences in mPPAR gamma 1 and mPPAR gamma 2 isoforms. The mPPAR gamma 1 and mPPAR gamma 2 isoforms are transcribed from different promoters. The mPPAR gamma gene has been mapped to chromosome 6 E3-F1 by in situ hybridization using a biotin-labeled probe. These results establish that at least one of the PPAR genes yields more than one protein product, similar to that encountered with retinoid X receptor and retinoic acid receptor genes. The existence of multiple PPAR isoforms transcribed from different promoters could increase the diversity of ligand and tissue-specific transcriptional responses.

A mutated intron sequence codes for an antigenic peptide recognized by cytolytic T lymphocytes on a human melanoma.

We have identified an antigen recognized on a human melanoma by autologous cytolytic T lymphocytes. It is encoded by a gene that is expressed in many normal tissues. Remarkably, the sequence coding for the antigenic peptide is located across an exon-intron junction. A point mutation is present in the intron that generates an amino acid change that is essential for the recognition of the peptide by the anti-tumor cytotoxic T lymphocytes. This observation suggests that the T-cell-mediated surveillance of the integrity of the genome may extend to some intronic regions.

Tissue specificity of a glucocorticoid-dependent enhancer in transgenic mice.

The glucocorticoid-responsive units (GRUs) of the rat tyrosine aminotransferase were associated with the regulatory sequences of a cellular gene expressed ubiquitously--that coding for the largest subunit of RNA polymerase II. In transient expression assays, glucocorticoid responsiveness of the hybrid regulatory regions depends on the spatial relationship and number of regulatory elements. Two parameters affect the ratio of induction by glucocorticoids: the basal level of the hybrid promoter that is affected by the RNA polymerase II regulatory sequences and the glucocorticoid-induced level that depends on the distance between the GRUs and the TATA box. A fully active glucocorticoid-responsive hybrid gene was used to generate transgenic mice. Results show that a composite regulatory pattern is obtained: ubiquitous basal expression characteristic of the RNA polymerase II gene and liver-specific glucocorticoid activation characteristic of the tyrosine aminotransferase GRUs. This result demonstrates that the activity of the tyrosine aminotransferase GRUs is cell-type-specific not only in cultured cells but also in the whole animal.

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.

Increased mutation frequency of feline immunodeficiency virus lacking functional deoxyuridine-triphosphatase.

Feline immunodeficiency virus (FIV) encodes the enzyme deoxyuridine-triphosphatase (DU; EC 3.6.1.23) between the coding regions for reverse transcriptase and integrase in the pol gene. Here, we report the in vivo infection of cats with a DU- variant of the PPR strain of FIV and compare its growth properties and tissue distribution with those of wild-type FIV-PPR. The results reveal several important points: (i) DU- FIV is able to infect the cat, with kinetics similar to that observed with wild-type FIV; (ii) both wild-type and DU- FIV-infected specific-pathogen free cats mount a strong humoral antibody response which is able to limit the virus burden in both groups of animals; (iii) the virus burden is reduced in the DU- FIV-infected cats, particularly in tissues such as spleen and salivary gland; and (iv) the mutation frequency in DU- FIVs integrated in the DNA of primary macrophages after 9 months of infection is approximately 5-fold greater than the frequency observed in DU- FIV DNA integrated in T lymphocytes. Mutation rate with wild-type FIV remains the same in both cell types in vivo. The dominant mutations seen in macrophages with DU- FIV are G-->A base changes, consistent with an increased misincorporation of deoxyuridine into viral DNA of DU- FIVs during reverse transcription. Because this enzyme is absent from human immunodeficiency virus type 1 and other primate lentiviruses, virus replication in cell environments with low DU activity may lead to increased mutation and contribute to the rapid expansion of the viral repertoire.

Structure of the gene for porcine peptide antibiotic PR-39, a cathelin gene family member: comparative mapping of the locus for the human peptide antibiotic FALL-39.

PR-39 is a porcine 39-aa peptide antibiotic composed of 49% proline and 24% arginine, with an activity against Gram-negative bacteria comparable to that of tetracycline. In Escherichia coli, it inhibits DNA and protein synthesis. PR-39 was originally isolated from pig small intestine, but subsequent cDNA cloning showed that the gene is expressed in the bone marrow. The open reading frame of the clone showed that PR-39 is made as 173-aa precursor whose proregion belongs to the cathelin family. The PR39 gene, which is rather compact and spans only 1784 bp has now been sequenced. The coding information is split into four exons. The first exon contains the signal sequence of 29 residues and the first 37 residues of the cathelin propart. Exons 2 and 3 contain only cathelin information, while exon 4 codes for the four C-terminal cathelin residues and the mature PR-39 peptide extended by three residues. The sequenced upstream region (1183 bp) contains four potential recognition sites for NF-IL6 and three for APRF, transcription factors known to regulate genes for both cytokines and acute phase response factors. Genomic hybridizations revealed a fairly high level of restriction fragment length polymorphism and indicated that there are at least two copies of the PR39 gene in the pig genome. PR39 was mapped to pig chromosome 13 by linkage and in situ hybridization mapping. The gene for the human peptide antibiotic FALL-39 (also a member of the cathelin family) was mapped to human chromosome 3, which is homologous to pig chromosome 13.

The choice of alternative 5' splice sites in influenza virus M1 mRNA is regulated by the viral polymerase complex.

The influenza virus M1 mRNA has two alternative 5' splice sites: a distal 5' splice site producing mRNA3 that has the coding potential for 9 amino acids and a proximal 5' splice site producing M2 mRNA encoding the essential M2 ion-channel protein. Only mRNA3 was made in uninfected cells transfected with DNA expressing M1 mRNA. Similarly, using nuclear extracts from uninfected cells, in vitro splicing of M1 mRNA yielded only mRNA3. Only when the mRNA3 5' splice site was inactivated by mutation was M2 mRNA made in uninfected cells and in uninfected cell extracts. In influenza virus-infected cells, M2 mRNA was made, but only after a delay, suggesting that newly synthesized viral gene product(s) were needed to activate the M2 5' splice site. We present strong evidence that these gene products are the complex of the three polymerase proteins, the same complex that functions in the transcription and replication of the viral genome. Gel shift experiments showed that the viral polymerase complex bound to the 5' end of the viral M1 mRNA in a sequence-specific and cap-dependent manner. During in vitro splicing catalyzed by uninfected cell extracts, the binding of the viral polymerase complex blocked the mRNA3 5' splice site, resulting in the switch to the M2 mRNA 5' splice site and the production of M2 mRNA.

Complete congruence between gene diversity estimates derived from genotypic data at enzyme and random amplified polymorphic DNA loci in black spruce.

Controversy still exists over the adaptive nature of variation of enzyme loci. In conifers, random amplified polymorphic DNAs (RAPDs) represent a class of marker loci that is unlikely to fall within or be strongly linked to coding DNA. We have compared the genetic diversity in natural populations of black spruce [Picea mariana (Mill.) B.S.P.] using genotypic data at allozyme loci and RAPD loci as well as phenotypic data from inferred RAPD fingerprints. The genotypic data for both allozymes and RAPDs were obtained from at least six haploid megagametophytes for each of 75 sexually mature individuals distributed in five populations. Heterozygosities and population fixation indices were in complete agreement between allozyme loci and RAPD loci. In black spruce, it is more likely that the similar levels of variation detected at both enzyme and RAPD loci are due to such evolutionary forces as migration and the mating system, rather than to balancing selection and overdominance. Furthermore, we show that biased estimates of expected heterozygosity and among-population differentiation are obtained when using allele frequencies derived from dominant RAPD phenotypes.

The human plakoglobin gene localizes on chromosome 17q21 and is subjected to loss of heterozygosity in breast and ovarian cancers.

The gene encoding human plakoglobin was mapped to chromosome 17q12-q22. An intragenic restriction fragment length polymorphism was used to localize the plakoglobin gene distal to locus KRT10 and proximal to the marker D17S858. The plakoglobin gene colocalizes with the polymorphic 17q21 marker UM8 on the same cosmid insert. This subregion of chromosome 17 is known to be particularly subjected to genetic alterations in sporadic breast and ovarian tumors. We show loss of heterozygosity of the plakoglobin gene in breast and ovarian tumors. We have identified a low-frequency polymorphism in the plakoglobin coding sequence which results in an arginine to histidine substitution at amino acid position 142 of the protein, as well as a silent mutation at nucleotide position 332 of the coding sequence. This polymorphism allowed us to demonstrate an allelic association of plakoglobin with predisposition to familial breast and ovarian cancers. Our results, together with the present knowledge about the biological function of plakoglobin, suggest that plakoglobin might represent a putative tumor suppressor gene for breast and ovarian cancers.

Construction of a 2.8-megabase yeast artificial chromosome contig and cloning of the human methylthioadenosine phosphorylase gene from the tumor suppressor region on 9p21.

Many human malignant cells lack methylthioadenosine phosphorylase (MTAP) enzyme activity. The gene (MTAP) encoding this enzyme was previously mapped to the short arm of chromosome 9, band p21-22, a region that is frequently deleted in multiple tumor types. To clone candidate tumor suppressor genes from the deleted region on 9p21-22, we have constructed a long-range physical map of 2.8 megabases for 9p21 by using overlapping yeast artificial chromosome and cosmid clones. This map includes the type IIFN gene cluster, the recently identified candidate tumor suppressor genes CDKN2 (p16INK4A) and CDKN2B (p15INK4B), and several CpG islands. In addition, we have identified other transcription units within the yeast artificial chromosome contig. Sequence analysis of a 2.5-kb cDNA clone isolated from a CpG island that maps between the IFN genes and CDKN2 reveals a predicted open reading frame of 283 amino acids followed by 1302 nucleotides of 3' untranslated sequence. This gene is evolutionarily conserved and shows significant amino acid homologies to mouse and human purine nucleoside phosphorylases and to a hypothetical 25.8-kDa protein in the pet gene (coding for cytochrome bc1 complex) region of Rhodospirillum rubrum. The location, expression pattern, and nucleotide sequence of this gene suggest that it codes for the MTAP enzyme.