Genetic mechanisms of Na+, K+-ATPase regulation in hypertension

The spontaneously hypertensive rat (SHR) is a model of essential hypertension. During the early development of hypertension, the SHR demonstrates increased proximal tubule (PT) Na+ reabsorption. I hypothesized that the increased PT Na+ reabsorption exhibited by the young SHR was due to altered sub-cellular distribution of Na+, K +-ATPase compared to the normotensive Wistar Kyoto (WKY). The hypothesis is supported, herein, by observations of greater Na+, K +-ATPase α 1 abundance in PT plasma membrane and lower abundance in late endosomes of 4wk SHR despite no difference in total PT α 1 abundance. There is a greater amount of Ser-18 unphosphorylated α 1 in the 4wk SHR PT. Total PT Na+, K+-ATPase γ abundance is greater in SHR at 4wk and 16wk but γ abundance in plasma membrane is greater only at 4wk. The phosphatase, calcineurin, was chosen for study because it is involved in the stimulation of Na+, K +-ATPase. No difference in calcineurin coding sequence, expression, or activity was observed in SHR. Gene expression arrays were next used to find candidate genes involved in the regulation of Na+, K +-ATPase. The first candidate analyzed was soluble epoxide hydrolase (sEH). The gene encoding sEH (EPHX2) showed lower expression in SHR. There was also a reduction in sEH protein abundance but there was no correlation between protein abundance and blood pressure in F2 progeny. Two EPHX2 alleles were identified, an ancestral allele and a variant allele containing four polymorphisms. sEH activity was greater in animals carrying the variant allele but the inheritance of the variant allele did not correlate with blood pressure. Gene expression arrays also led to the examination of genes involved in redox balance/Na+, K+-ATPase regulation. A pattern of lower expression of genes involved in reactive radical detoxification in SHR was discerned. Six transcription factor binding sites were identified that occurred more often in these genes. Three transcription factors that bind to the HNF1 site were expressed at lower levels in SHR. This points to the HNF1 transcriptional complex as an important trans-acting regulator of a wide range of genes involved in altered redox balance in SHR. ^

Herpes simplex virus vector-mediated expression of Bcl-2 prevents 6-hydroxydopamine-induced degeneration of neurons in the substantia nigra in vivo

6-Hydroxydopamine (6-OHDA) is widely used to selectively lesion dopaminergic neurons of the substantia nigra (SN) in the creation of animal models of Parkinson’s disease. In vitro, the death of PC-12 cells caused by exposure to 6-OHDA occurs with characteristics consistent with an apoptotic mechanism of cell death. To test the hypothesis that apoptotic pathways are involved in the death of dopaminergic neurons of the SN caused by 6-OHDA, we created a replication-defective genomic herpes simplex virus-based vector containing the coding sequence for the antiapoptotic peptide Bcl-2 under the transcriptional control of the simian cytomegalovirus immediate early promoter. Transfection of primary cortical neurons in culture with the Bcl-2-producing vector protected those cells from naturally occurring cell death over 3 weeks. Injection of the Bcl-2-expressing vector into SN of rats 1 week before injection of 6-OHDA into the ipsilateral striatum increased the survival of neurons in the SN, detected either by retrograde labeling of those cells with fluorogold or by tyrosine hydroxylase immunocytochemistry, by 50%. These results, demonstrating that death of nigral neurons induced by 6-OHDA lesioning may be blocked by the expression of Bcl-2, are consistent with the notion that cell death in this model system is at least in part apoptotic in nature and suggest that a Bcl-2-expressing vector may have therapeutic potential in the treatment of Parkinson’s disease.

Amyrel, a paralogous gene of the amylase gene family in Drosophila melanogaster and the Sophophora subgenus

We describe a gene from Drosophila melanogaster related to the alpha-amylase gene Amy. This gene, which exists as a single copy, was named Amyrel. It is strikingly divergent from Amy because the amino acid divergence is 40%. The coding sequence is interrupted by a short intron at position 655, which is unusual in amylase genes. Amyrel has also been cloned in Drosophila ananassae, Drosophila pseudoobscura, and Drosophila subobscura and is likely to be present throughout the Sophophora subgenus, but, to our knowledge, it has not been detected outside. Unexpectedly, there is a strong conservation of 5′ and 3′ flanking regions between Amyrel genes from different species, which is not the case for Amy and which suggests that selection acts on these regions. In contrast to the Amy genes, Amyrel is transcribed in larvae of D. melanogaster but not in adults. However, the protein has not been detected yet. Amyrel evolves about twice as fast as Amy in the several species studied. We suggest that this gene could result from a duplication of Amy followed by accelerated and selected divergence toward a new adaptation.

Chlorophyll a oxygenase (CAO) is involved in chlorophyll b formation from chlorophyll a

Chlorophyll b is an ubiquitous accessory pigment in land plants, green algae, and prochlorophytes. Its biosynthesis plays a key role in the adaptation to various light environments. We isolated six chlorophyll b-less mutants by insertional mutagenesis by using the nitrate reductase or argininosuccinate lyase genes as tags and examined the rearrangement of mutant genomes. We found that an overlapping region of a nuclear genome was deleted in all mutants and that this encodes a protein whose sequence is similar to those of methyl monooxygenases. This coding sequence also contains putative binding domains for a [2Fe-2S] Rieske center and for a mononuclear iron. The results demonstrate that a chlorophyll a oxygenase is involved in chlorophyll b formation. The reaction mechanism of chlorophyll b formation is discussed.

A variant of DNA polymerase β acts as a dominant negative mutant

In eukaryotic cells, DNA polymerase β (polβ) carries out base-excision repair (BER) required for DNA maintenance, replication, recombination, and drug resistance. A specific deletion in one allele in the coding sequence of the polβ gene occurs in colorectal and breast carcinomas. The 87-bp deleted region encodes amino acid residues 208–236 in the catalytic domain of the enzyme. Here, we report evidence for expression of the wild-type (WT) and the truncated polβ proteins in colorectal tumors. To elucidate the potential functional consequences of polβ truncation, stable HeLa cell lines were established from cloned WT and variant polβΔ208–236. Cells expressing the variant protein exhibited substantially decreased BER activity. To test our hypothesis that truncated polβ may disrupt the function of the WT enzyme, we stably transfected mouse embryonic fibroblast 16.3 cells with polβΔ208–236 cDNA. Reverse transcription–PCR and Western blot analyses showed that the new cell line, 16.3ΔP, expresses the WT and the truncated polβ mRNA and proteins. BER and binding activities were undetectable in these cells. Furthermore, in vivo the 16.3ΔP cells were more sensitive to N-methyl-N′-nitro-N-nitrosoguanidine than the 16.3 cells. On adding increasing amounts of 16.3ΔP protein extracts, the BER and DNA binding activities of extracts of the parent 16.3 cell line progressively declined. These results strongly suggest that truncated polβ acts as a dominant negative mutant. The defective polβ may facilitate accumulation of mutations, leading to the expression of a mutator phenotype in tumor cells.

β-Catenin mutations in cell lines established from human colorectal cancers

β-catenin has functions as both an adhesion and a signaling molecule. Disruption of these functions through mutations of the β-catenin gene (CTNNB1) may be important in the development of colorectal tumors. We examined the entire coding sequence of β-catenin by reverse transcriptase–PCR (RT-PCR) and direct sequencing of 23 human colorectal cancer cell lines from 21 patients. In two cell lines, there was apparent instability of the β-catenin mRNA. Five different mutations (26%) were found in the remaining 21cell lines (from 19 patients). A three-base deletion (codon 45) was identified in the cell line HCT 116, whereas cell lines SW 48, HCA 46, CACO 2, and Colo 201 each contained single-base missense mutations (codons 33, 183, 245, and 287, respectively). All 23 cell lines had full-length β-catenin protein that was detectable by Western blotting and that coprecipitated with E-cadherin. In three of the cell lines with CTNNB1 mutations, complexes of β-catenin with α-catenin and APC were detectable. In SW48 and HCA 46, however, we did not detect complexes of β-catenin protein with α-catenin and APC, respectively. These results show that selection of CTNNB1 mutations occurs in up to 26% of colorectal cancers from which cell lines are derived. In these cases, mutation selection is probably for altered β-catenin function, which may significantly alter intracellular signaling and intercellular adhesion and may serve as a complement to APC mutations in the early stages of tumorigenesis.

A viral suppressor of gene silencing in plants

Gene silencing is an important but little understood regulatory mechanism in plants. Here we report that a viral sequence, initially identified as a mediator of synergistic viral disease, acts to suppress the establishment of both transgene-induced and virus-induced posttranscriptional gene silencing. The viral suppressor of silencing comprises the 5′-proximal region of the tobacco etch potyviral genomic RNA encoding P1, helper component-proteinase (HC-Pro) and a small part of P3, and is termed the P1/HC-Pro sequence. A reversal of silencing assay was used to assess the effect of the P1/HC-Pro sequence on transgenic tobacco plants (line T4) that are posttranscriptionally silenced for the uidA reporter gene. Silencing was lifted in offspring of T4 crosses with four independent transgenic lines expressing P1/HC-Pro, but not in offspring of control crosses. Viral vectors were used to assess the effect of P1/HC-Pro expression on virus-induced gene silencing (VIGS). The ability of a potato virus X vector expressing green fluorescent protein to induce silencing of a green fluorescent protein transgene was eliminated or greatly reduced when P1/HC-Pro was expressed from the same vector or from coinfecting potato virus X vectors. Expression of the HC-Pro coding sequence alone was sufficient to suppress virus-induced gene silencing, and the HC-Pro protein product was required for the suppression. This discovery points to the role of gene silencing as a natural antiviral defense system in plants and offers different approaches to elucidate the molecular basis of gene silencing.

Sulfate reduction in higher plants: Molecular evidence for a novel 5′-adenylylsulfate reductase

Sulfate-assimilating organisms reduce inorganic sulfate for Cys biosynthesis. There are two leading hypotheses for the mechanism of sulfate reduction in higher plants. In one, adenosine 5′-phosphosulfate (APS) (5′-adenylylsulfate) sulfotransferase carries out reductive transfer of sulfate from APS to reduced glutathione. Alternatively, the mechanism may be similar to that in bacteria in which the enzyme, 3′-phosphoadenosine-5′-phosphosulfate (PAPS) reductase, catalyzes thioredoxin (Trx)-dependent reduction of PAPS. Three classes of cDNA were cloned from Arabidopsis thaliana termed APR1, -2, and -3, that functionally complement a cysH, PAPS reductase mutant strain of Escherichia coli. The coding sequence of the APR clones is homologous with PAPS reductases from microorganisms. In addition, a carboxyl-terminal domain is homologous with members of the Trx superfamily. Further genetic analysis showed that the APR clones can functionally complement a mutant strain of E. coli lacking Trx, and an APS kinase, cysC. mutant. These results suggest that the APR enzyme may be a Trx-independent APS reductase. Cell extracts of E. coli expressing APR showed Trx-independent sulfonucleotide reductase activity with a preference for APS over PAPS as a substrate. APR-mediated APS reduction is dependent on dithiothreitol, has a pH optimum of 8.5, is stimulated by high ionic strength, and is sensitive to inactivation by 5′-adenosinemonophosphate (5′-AMP). 2′-AMP, or 3′-phosphoadenosine-5′-phosphate (PAP), a competitive inhibitor of PAPS reductase, do not affect activity. The APR enzymes may be localized in different cellular compartments as evidenced by the presence of an amino-terminal transit peptide for plastid localization in APR1 and APR3 but not APR2. Southern blot analysis confirmed that the APR clones are members of a small gene family, possibly consisting of three members.

Tsetse thrombin inhibitor: Bloodmeal-induced expression of an anticoagulant in salivary glands and gut tissue of Glossina morsitans morsitans

The tsetse thrombin inhibitor, a potent and specific low molecular mass (3,530 Da) anticoagulant peptide, was purified previously from salivary gland extracts of Glossina morsitans morsitans (Diptera: Glossinidae). A 303-bp coding sequence corresponding to the inhibitor has now been isolated from a tsetse salivary gland cDNA library by using degenerate oligonucleotide probes. The full-length cDNA contains a 26-bp untranslated segment at its 5′ end, followed by a 63-bp sequence corresponding to a putative secretory signal peptide. A 96-bp segment codes for the mature tsetse thrombin inhibitor, whose predicted molecular weight matches that of the purified native protein. Based on its lack of homology to any previously described family of molecules, the tsetse thrombin inhibitor appears to represent a unique class of naturally occurring protease inhibitors. Recombinant tsetse thrombin inhibitor expressed in Escherichia coli and the chemically synthesized peptide are both substantially less active than the purified native protein, suggesting that posttranslational modification(s) may be necessary for optimal inhibitory activity. The tsetse thrombin inhibitor gene, which is present as a single copy in the tsetse genome, is expressed at high levels in salivary glands and midguts of adult tsetse flies, suggesting a possible role for the anticoagulant in both feeding and processing of the bloodmeal.

The increased level of β1,4-galactosyltransferase required for lactose biosynthesis is achieved in part by translational control

β1,4-Galactosyltransferase (β4GalT-I) participates in both glycoconjugate biosynthesis (ubiquitous activity) and lactose biosynthesis (mammary gland-specific activity). In somatic tissues, transcription of the mammalian β4GalT-I gene results in a 4.1-kb mRNA and a 3.9-kb mRNA as a consequence of initiation at two start sites separated by ≈200 bp. In the mammary gland, coincident with the increased β4GalT-I enzyme level (≈50-fold) required for lactose biosynthesis, there is a switch from the 4.1-kb start site to the preferential use of the 3.9-kb start site, which is governed by a stronger tissue-restricted promoter. The use of the 3.9-kb start site results in a β4GalT-I transcript in which the 5′- untranslated region (UTR) has been truncated from ≈175 nt to ≈28 nt. The 5′-UTR of the 4.1-kb transcript [UTR(4.1)] is predicted to contain extensive secondary structure, a feature previously shown to reduce translational efficiency of an mRNA. In contrast, the 5′-UTR of the 3.9-kb mRNA [UTR(3.9)] lacks extensive secondary structure; thus, this transcript is predicted to be more efficiently translated relative to the 4.1-kb mRNA. To test this prediction, constructs were assembled in which the respective 5′-UTRs were fused to the luciferase-coding sequence and enzyme levels were determined after translation in vitro and in vivo. The luciferase mRNA containing the truncated UTR(3.9) was translated more efficiently both in vitro (≈14-fold) and in vivo (3- to 5-fold) relative to the luciferase mRNA containing the UTR(4.1). Consequently, in addition to control at the transcriptional level, β4GalT-I enzyme levels are further augmented in the lactating mammary gland as a result of translational control.

λ Rap protein is a structure-specific endonuclease involved in phage recombination

Bacteriophage λ encodes a number of genes involved in the recombinational repair of DNA double-strand breaks. The product of one of these genes, rap, has been purified. Truncated Rap proteins that copurify with the full-length form are derived, at least in part, from a ρ-dependent transcription terminator located within its coding sequence. Full-length and certain truncated Rap polypeptides bind preferentially to branched DNA substrates, including synthetic Holliday junctions and D-loops. In the presence of manganese ions, Rap acts as an endonuclease that cleaves at the branch point of Holliday and D-loop substrates. It shows no obvious sequence preference or symmetry of cleavage on a Holliday junction. The biochemical analysis of Rap gives an insight into how recombinants could be generated by the nicking of a D-loop without the formation of a classical Holliday junction.

A model for spontaneous B-lineage lymphomas in IgHμ-HOX11 transgenic mice

HOX11, a divergent homeodomain-containing transcription factor, was isolated from the breakpoint of the nonrandom t(10;14)(q24;q11) chromosome translocation found in human T cell acute lymphoblastic leukemias. The translocation places the HOX11 coding sequence under the transcriptional control of TCR α/δ regulatory elements, resulting in ectopic expression of a normal HOX11 protein in thymocytes. To investigate the oncogenic potential of HOX11, we targeted its expression in lymphocytes of transgenic mice by placing the human cellular DNA under the transcriptional control of Ig heavy chain or LCK regulatory sequences. Only IgHμ-HOX11 mice expressing low levels of HOX11 were viable. During their second year of life, all HOX11 transgenic mice became terminally ill with more than 75% developing large cell lymphomas in the spleen, which frequently disseminated to thymus, lymph nodes, and other nonhematopoietic tissues. Lymphoma cells were predominantly clonal IgM+IgD+ mature B cells. Repopulation of severe combined immunodeficient mice with cells from hyperplastic spleens indicated that the HOX11 tumor phenotype was transplantable. Before tumor development, expression of the transgene did not result in perturbations in lymphopoiesis; however, lymphoid hyperplasia involving the splenic marginal zones was present in 20% of spleens. Our studies provide direct evidence that expression of HOX11 in lymphocytes leads to malignant transformation. These mice are a useful model system to study mechanisms involved in transformation from B-lineage hyperplasia to malignant lymphoma and for testing novel approaches to therapy. They represent a novel animal model for non-Hodgkin’s lymphoma of peripheral mature B cell origin.

Double muscling in cattle due to mutations in the myostatin gene

Myostatin (GDF-8) is a member of the transforming growth factor β superfamily of secreted growth and differentiation factors that is essential for proper regulation of skeletal muscle mass in mice. Here we report the myostatin sequences of nine other vertebrate species and the identification of mutations in the coding sequence of bovine myostatin in two breeds of double-muscled cattle, Belgian Blue and Piedmontese, which are known to have an increase in muscle mass relative to conventional cattle. The Belgian Blue myostatin sequence contains an 11-nucleotide deletion in the third exon which causes a frameshift that eliminates virtually all of the mature, active region of the molecule. The Piedmontese myostatin sequence contains a missense mutation in exon 3, resulting in a substitution of tyrosine for an invariant cysteine in the mature region of the protein. The similarity in phenotypes of double-muscled cattle and myostatin null mice suggests that myostatin performs the same biological function in these two species and is a potentially useful target for genetic manipulation in other farm animals.

Inhibition of ubiquitin/proteasome-dependent protein degradation by the Gly-Ala repeat domain of the Epstein–Barr virus nuclear antigen 1

The Epstein–Barr virus (EBV) encoded nuclear antigen (EBNA) 1 is expressed in latently infected B lymphocytes that persist for life in healthy virus carriers and is the only viral protein regularly detected in all EBV associated malignancies. The Gly-Ala repeat domain of EBNA1 was shown to inhibit in cis the presentation of major histocompatibility complex (MHC) class I restricted cytotoxic T cell epitopes from EBNA4. It appears that the majority of antigens presented via the MHC I pathway are subject to ATP-dependent ubiquitination and degradation by the proteasome. We have investigated the influence of the repeat on this process by comparing the degradation of EBNA1, EBNA4, and Gly-Ala containing EBNA4 chimeras in a cell-free system. EBNA4 was efficiently degraded in an ATP/ubiquitin/proteasome-dependent fashion whereas EBNA1 was resistant to degradation. Processing of EBNA1 was restored by deletion of the Gly-Ala domain whereas insertion of Gly-Ala repeats of various lengths and in different positions prevented the degradation of EBNA4 without appreciable effect on ubiquitination. Inhibition was also achieved by insertion of a Pro-Ala coding sequence. The results suggest that the repeat may affect MHC I restricted responses by inhibiting antigen processing via the ubiquitin/proteasome pathway. The presence of regularly interspersed Ala residues appears to be important for the effect.

Protamine-Cre recombinase transgenes efficiently recombine target sequences in the male germ line of mice, but not in embryonic stem cells

The production of subtle or conditional mutations in mice through the combined use of site-specific and homologous recombination has become an increasingly widespread experimental paradigm in mammalian genetics. Embryonic stem cells containing recombinase transgenes that were expressed in the male germ line, but not in other tissues or in the embryonic stem cells themselves, would substantially simplify the production of such alleles. Here we show that transgenes comprised of the mouse protamine 1 promoter and the Cre recombinase coding sequence mediate the efficient recombination of a Cre target transgene in the male germ line, but not in other tissues. Embryonic stem cell lines generated from one of these transgenic strains were transfected with targeting vectors that included loxP-flanked selectable markers, and homologously recombined alleles containing the marker and functional loxP sites were isolated. These results establish the potential of the system for substantially reducing the time, effort, and resources required to produce homologously recombined alleles in mice that have been secondarily rearranged by a site-specific recombinase.