Characterization of the gene cluster of high-molecular-mass nitrile hydratase (H-NHase) induced by its reaction product in Rhodococcus rhodochrous J1.

The 4.6-kb region 5'-upstream from the gene encoding a cobalt-containing and amide-induced high molecular mass-nitrile hydratase (H-NHase) from Rhodococcus rhodochrous J1 was found to be required for the expression of the H-NHase gene with a host-vector system in a Rhodococcus strain. Sequence analysis has revealed that there are at least five open reading frames (H-ORF1 approximately 5) in addition to H-NHase alpha- and beta-subunit genes. Deletion of H-ORF1 and H-ORF2 resulted in decrease of NHase activity, suggesting a positive regulatory role of both ORFs in the expression of the H-NHase gene. H-ORF1 showed significant similarity to a regulatory protein, AmiC, which is involved in regulation of amidase expression by binding an inducer amide in Pseudomonas aeruginosa. H-ORF4, which has been found to be uninvolved in regulation of H-NHase expression by enzyme assay for its deletion transformant and Northern blot analysis for R. rhodochrous J1, showed high similarity to transposases from insertion sequences of several bacteria. Determination of H-NHase activity and H-NHase mRNA levels in R. rhodochrous J1 has indicated that the expression of the H-NHase gene is regulated by an amide at the transcriptional level. These findings suggest the participation of H-ORF4 (IS1164) in the organization of the H-NHase gene cluster and the involvement of H-ORF1 in unusual induction mechanism, in which H-NHase is formed by amides (the products in the NHase reaction), but not by nitriles (the substrates).

The product of the ataxia-telangiectasia group D complementing gene, ATDC, interacts with a protein kinase C substrate and inhibitor.

Ataxia-telangiectasia (AT) is an autosomal recessive human genetic disease characterized by immunological, neurological, and developmental defects and an increased risk of cancer. Cells from individuals with AT show sensitivity to ionizing radiation, elevated recombination, cell cycle abnormalities, and aberrant cytoskeletal organization. The molecular basis of the defect is unknown. A candidate AT gene (ATDC) was isolated on the basis of its ability to complement the ionizing radiation sensitivity of AT group D fibroblasts. Whether ATDC is mutated in any AT patients is not known. We have found that the ATDC protein physically interacts with the intermediate-filament protein vimentin, which is a protein kinase C substrate and colocalizing protein, and with an inhibitor of protein kinase C, hPKCI-1. Indirect immunofluorescence analysis of cultured cells transfected with a plasmid encoding an epitope-tagged ATDC protein localizes the protein to vimentin filaments. We suggest that the ATDC and hPKCI-1 proteins may be components of a signal transduction pathway that is induced by ionizing radiation and mediated by protein kinase C.

Characterization of mice deficient in aromatase (ArKO) because of targeted disruption of the cyp19 gene

The formation of estrogens from C19 steroids is catalyzed by aromatase cytochrome P450 (P450arom), the product of the cyp19 gene. The actions of estrogen include dimorphic anatomical, functional, and behavioral effects on the development of both males and females, considerations that prompted us to examine the consequences of deficiency of aromatase activity in mice. Mice lacking a functional aromatase enzyme (ArKO) were generated by targeted disruption of the cyp19 gene. Male and female ArKO mice were born with the expected Mendelian frequency from F1 parents and grew to adulthood. Female ArKO mice at 9 weeks of age displayed underdeveloped external genitalia and uteri. Ovaries contained numerous follicles with abundant granulosa cells and evidence of antrum formation that appeared arrested before ovulation. No corpora lutea were present. Additionally the stroma were hyperplastic with structures that appeared to be atretic follicles. Development of the mammary glands approximated that of a prepubertal female. Examination of male ArKO mice of the same age revealed essentially normal internal anatomy but with enlargement of the male accessory sex glands because of increased content of secreted material. The testes appeared normal. Male ArKO mice are capable of breeding and produce litters of approximately average size. Whereas serum estradiol levels were at the limit of detection, testosterone levels were elevated, as were the levels of follicle-stimulating hormone and luteinizing hormone. The phenotype of these animals differs markedly from that of the previously reported ERKO mice, in which the estrogen receptor α is deleted by targeted disruption.

Chymase cleavage of stem cell factor yields a bioactive, soluble product

Stem cell factor (SCF) is produced by stromal cells as a membrane-bound molecule, which may be proteolytically cleaved at a site close to the membrane to produce a soluble bioactive form. The proteases producing this cleavage are unknown. In this study, we demonstrate that human mast cell chymase, a chymotrypsin-like protease, cleaves SCF at a novel site. Cleavage is at the peptide bond between Phe-158 and Met-159, which are encoded by exon 6 of the SCF gene. This cleavage results in a soluble bioactive product that is 7 amino acids shorter at the C terminus than previously identified soluble SCF. This research shows the identification of a physiologically relevant enzyme that specifically cleaves SCF. Because mast cells express the KIT protein, the receptor for SCF, and respond to SCF by proliferation and degranulation, this observation identifies a possible feedback loop in which chymase released from mast cell secretory granules may solubilize SCF bound to the membrane of surrounding stromal cells. The liberated soluble SCF may in turn stimulate mast cell proliferation and differentiated functions; this loop could contribute to abnormal accumulations of mast cells in the skin and hyperpigmentation at sites of chronic cutaneous inflammation.

Characterization of the Saccharomyces cerevisiae ERG27 gene encoding the 3-keto reductase involved in C-4 sterol demethylation

The last unidentified gene encoding an enzyme involved in ergosterol biosynthesis in Saccharomyces cerevisiae has been cloned. This gene, designated ERG27, encodes the 3-keto sterol reductase, which, in concert with the C-4 sterol methyloxidase (ERG25) and the C-3 sterol dehydrogenase (ERG26), catalyzes the sequential removal of the two methyl groups at the sterol C-4 position. We developed a strategy to isolate a mutant deficient in converting 3-keto to 3-hydroxy-sterols. An ergosterol auxotroph unable to synthesize sterol or grow without sterol supplementation was mutagenized. Colonies were then selected that were nystatin-resistant in the presence of 3-ketoergostadiene and cholesterol. A new ergosterol auxotroph unable to grow on 3-ketosterols without the addition of cholesterol was isolated. The gene (YLR100w) was identified by complementation. Segregants containing the YLR100w disruption failed to grow on various types of 3-keto sterol substrates. Surprisingly, when erg27 was grown on cholesterol- or ergosterol-supplemented media, the endogenous compounds that accumulated were noncyclic sterol intermediates (squalene, squalene epoxide, and squalene dioxide), and there was little or no accumulation of lanosterol or 3-ketosterols. Feeding experiments in which erg27 strains were supplemented with lanosterol (an upstream intermediate of the C-4 demethylation process) and cholesterol (an end-product sterol) demonstrated accumulation of four types of 3-keto sterols identified by GC/MS and chromatographic properties: 4-methyl-zymosterone, zymosterone, 4-methyl-fecosterone, and ergosta-7,24 (28)-dien-3-one. In addition, a fifth intermediate was isolated and identified by 1H NMR as a 4-methyl-24,25-epoxy-cholesta-7-en-3-one. Implications of these results are discussed.

Screening for imprinted genes by allelic message display: Identification of a paternally expressed gene Impact on mouse chromosome 18

A systematic screen termed the allelic message display (AMD) was developed for the hunting of imprinted genes. In AMD, differential display PCR is adopted to image allelic expression status of multiple polymorphic transcripts in two parental mouse strains, reciprocal F1 hybrids and pooled backcross progenies. From the displayed patterns, paternally and maternally expressed transcripts can be unequivocally identified. The effectiveness of AMD screening was clearly demonstrated by the identification of a paternally expressed gene Impact on mouse chromosome 18, the predicted product of which belongs to the YCR59c/yigZ hypothetical protein family composed of yeast and bacterial proteins with currently unknown function. In contrast with previous screening methods necessitating positional cloning efforts or generation of parthenogenetic embryos, this approach requires nothing particular but appropriately crossed mice and can be readily applied to any tissues at various developmental stages. Hence, AMD would considerably accelerate the identification of imprinted genes playing pivotal roles in mammalian development and the pathogenesis of various diseases.

Mendel’s dwarfing gene: cDNAs from the Le alleles and function of the expressed proteins

The major gibberellin (GA) controlling stem elongation in pea (Pisum sativum L.) is GA1, which is formed from GA20 by 3β-hydroxylation. This step, which limits GA1 biosynthesis in pea, is controlled by the Le locus, one of the original Mendelian loci. Mutations in this locus result in dwarfism. We have isolated cDNAs encoding a GA 3β-hydroxylase from lines of pea carrying the Le, le, le-3, and led alleles. The cDNA sequences from le and le-3 each contain a base substitution resulting in single amino acid changes relative to the sequence from Le. The cDNA sequence from led, a mutant derived from an le line, contains both the le “mutation” and a single-base deletion, which causes a shift in reading frame and presumably a null mutation. cDNAs from each line were expressed in Escherichia coli. The expression product for the clone from Le converted GA9 to GA4, and GA20 to GA1, with Km values of 1.5 μM and 13 μM, respectively. The amino acid substitution in the clone from le increased Km for GA9 100-fold and reduced conversion of GA20 to almost nil. Expression products from le and le-3 possessed similar levels of 3β-hydroxylase activity, and the expression product from led was inactive. Our results suggest that the 3β-hydroxylase cDNA is encoded by Le. Le transcript is expressed in roots, shoots, and cotyledons of germinating pea seedlings, in internodes and leaves of established seedlings, and in developing seeds.

Transcriptional regulation of the Xlim-1 gene by activin is mediated by an element in intron I

The Xlim-1 gene is activated in the late blastula stage of Xenopus embryogenesis in the mesoderm, and its RNA product becomes concentrated in the Spemann organizer at early gastrula stage. A major regulator of early expression of Xlim-1 is activin or an activin-like signal. We report experiments aiming to identify the activin response element in the Xlim-1 gene. The 5′ flanking region of the gene contains a constitutive promoter that is not activin responsive, whereas sequences in the first intron mediate repression of basal promoter activity and stimulation by activin. An intron-derived fragment of 212 nt is the smallest element that could mediate activin responsiveness. Nodal and act-Vg1, factors with signaling properties similar to activin, also stimulated Xlim-1 reporter constructs, whereas BMP-4 did not stimulate or repress the constructs. The mechanism of activin regulation of Xlim-1 and the sequence of the response element are distinct from activin response elements of other genes studied so far.

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.

A reversibly glycosylated polypeptide (RGP1) possibly involved in plant cell wall synthesis: Purification, gene cloning, and trans-Golgi localization

We purified from pea (Pisum sativum) tissue an ≈40 kDa reversibly glycosylated polypeptide (RGP1) that can be glycosylated by UDP-Glc, UDP-Xyl, or UDP-Gal, and isolated a cDNA encoding it, apparently derived from a single-copy gene (Rgp1). Its predicted translation product has 364 aminoacyl residues and molecular mass of 41.5 kDa. RGP1 appears to be a membrane-peripheral protein. Immunogold labeling localizes it specifically to trans-Golgi dictyosomal cisternae. Along with other evidence, this suggests that RGP1 is involved in synthesis of xyloglucan and possibly other hemicelluloses. Corn (Zea mays) contains a biochemically similar and structurally homologous RGP1, which has been thought (it now seems mistakenly) to function in starch synthesis. The expressed sequence database also reveals close homologs of pea Rgp1 in Arabidopsis and rice (Oryza sativa). Rice possesses, in addition, a distinct but homologous sequence (Rgp2). RGP1 provides a polypeptide marker for Golgi membranes that should be useful in plant membrane studies.

Oryza sativa PSK gene encodes a precursor of phytosulfokine-α, a sulfated peptide growth factor found in plants

Phytosulfokine-α [PSK-α, Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln], a sulfated mitogenic peptide found in plants, strongly promotes proliferation of plant cells in culture at very low concentrations. Oryza sativa PSK (OsPSK) cDNA encoding a PSK-α precursor has been isolated. The cDNA is 725 base pairs long, and the 89-aa product, preprophytosulfokine, has a 22-aa hydrophobic region that resembles a cleavable leader peptide at its NH2 terminus. The PSK-α sequence occurs only once within the precursor, close to the COOH terminus. [Ser4]PSK-α was secreted by transgenic rice Oc cells harboring a mutated OsPSK cDNA, suggesting proteolytic processing from the larger precursor, a feature commonly found in animal systems. Whereas PSK-α in conditioned medium with sense transgenic Oc cells was 1.6 times as concentrated as in the control case, antisense transgenic Oc cells produced less than 60% of the control level. Preprophytosulfokine mRNA was detected at an elevated constitutive level in rice Oc culture cells on RNA blot analysis. Although PSK-α molecules have never been identified in any intact plant, reverse transcription–PCR analysis demonstrated that OsPSK is expressed in rice seedlings, indicating that PSK-α may be important for plant cell proliferation both in vitro and in vivo. DNA blot analysis demonstrated that OsPSK homologs may occur in dicot as well as monocot plants.

Soldier caste-specific gene expression in the mandibular glands of Hodotermopsis japonica (Isoptera: Termopsidae)

Although “polymorphic castes” in social insects are well known as one of the most important phenomena of polyphenism, few studies of caste-specific gene expressions have been performed in social insects. To identify genes specifically expressed in the soldier caste of the Japanese damp-wood termite Hodotermopsis japonica, we employed the differential-display method using oligo(dT) and arbitrary primers, compared mRNA from the heads of mature soldiers and pseudergates (worker caste), and identified a clone (PCR product) 329 bp in length termed SOL1. Northern blot analysis showed that the SOL1 mRNA is about 1.0 kb in length and is expressed specifically in mature soldiers, but not in pseudergates, even in the presoldier induction by juvenile hormone analogue, suggesting that the product is specific for terminally differentiated soldiers. By using the method of 5′- and 3′-rapid amplification of cDNA ends, we isolated the full length of SOL1 cDNA, which contained an ORF with a putative signal peptide at the N terminus. The sequence showed no significant homology with any other known protein sequences. In situ hybridization analysis showed that SOL1 is expressed specifically in the mandibular glands. These results strongly suggest that the SOL1 gene encodes a secretory protein specifically synthesized in the mandibular glands of the soldiers. Histological observations revealed that the gland actually develops during the differentiation into the soldier caste.

E2F4-RB and E2F4-p107 complexes suppress gene expression by transforming growth factor β through E2F binding sites

Transforming growth factor β (TGF-β) causes growth arrest in most cell types. TGF-β induces hypophosphorylation of retinoblastoma susceptibility gene 1 product (RB), which sequesters E2F factors needed for progression into S phase of the cell cycle, thereby leading to cell cycle arrest at G1. It is possible, however, that the E2F-RB complex induced by TGF-β may bind to E2F sites and suppress expression of specific genes whose promoters contain E2F binding sites. We show here that TGF-β treatment of HaCaT cells induced the formation of E2F4-RB and E2F4-p107 complexes, which are capable of binding to E2F sites. Disruption of their binding to DNA with mutation in the E2F sites did not change the expression from promoters of E2F1, B-myb, or HsORC1 genes in cycling HaCaT cells. However, the same mutation stimulated 5- to 6-fold higher expression from all three promoters in cells treated with TGF-β. These results suggest that E2F binding sites play an essential role in the transcription repression of these genes under TGF-β treatment. Consistent with their repression of TGF-β-induced gene expression, introduction of E2F sites into the promoter of cyclin-dependent kinase inhibitor p15INK4B gene effectively inhibited its induction by TGF-β. Experiments utilizing Gal4-RB and Gal4-p107 chimeric constructs demonstrated that either RB or p107 could directly repress TGF-β induction of p15INK4B gene when tethered to p15INK4B promoter through Gal4 DNA binding sites. Therefore, E2F functions to bring RB and p107 to E2F sites and represses gene expression by TGF-β. These results define a specific function for E2F4-RB and E2F4-p107 complexes in gene repression under TGF-β treatment, which may constitute an integral part of the TGF-β-induced growth arrest program.

The zntA gene of Escherichia coli encodes a Zn(II)-translocating P-type ATPase

The first Zn(II)-translocating P-type ATPase has been identified as the product of o732, a potential gene identified in the sequencing of the Escherichia coli genome. This gene, termed zntA, was disrupted by insertion of a kanamycin gene through homologous recombination. The mutant strain exhibited hypersensitivity to zinc and cadmium salts but not salts of other metals, suggesting a role in zinc homeostasis in E. coli. Everted membrane vesicles from a wild-type strain accumulated 65Zn(II) and 109Cd(II) by using ATP as an energy source. Transport was sensitive to vanadate, an inhibitor of P-type ATPases. Membrane vesicles from the zntA∷kan strain did not accumulate those metal ions. Both the sensitive phenotype and transport defect of the mutant were complemented by expression of zntA on a plasmid.

In vivo gene delivery to the liver using reconstituted chylomicron remnants as a novel nonviral vector

Lipoproteins are emulsion particles that consist of lipids and apolipoproteins. Their natural function is to transport lipids and/or cholesterol to different tissues. We have taken advantage of the hydrophobic interior of these natural emulsions to solubilize DNA. Negatively charged DNA was first complexed with cationic lipids containing a quaternary amine head group. The resulting hydrophobic complex was extracted by chloroform and then incorporated into reconstituted chylomicron remnant particles (≈100 nm in diameter) with an efficiency ≈65%. When injected into the portal vein of mice, there were ≈5 ng of a transgene product (luciferase) produced per mg of liver protein per 100 μg injected DNA. This level of transgene expression was ≈100-fold higher than that of mice injected with naked DNA. However, such a high expression was not found after tail vein injection. Histochemical examination revealed that a large number of parenchymal cells and other types of cells in the liver expressed the transgene. Gene expression in the liver increased with increasing injected dose, and was nearly saturated with 50 μg DNA. At this dose, the expression was kept at high level in the liver for 2 days and then gradually reduced and almost disappeared by 7 days. However, by additional injection at day 7, gene expression in the liver was completely restored. By injection of plasmid DNA encoding human α1-antitrypsin, significant concentrations of hAAT were detected in the serum of injected animals. This is the first nonviral vector that resembles a natural lipoprotein carrier.