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The isolation, characterization, and molecular cloning of a 75-kDa gelatinase B-like enzyme, a member of the matrix metalloproteinase (MMP) family. An avian enzyme that is MMP-9-like in its cell expression pattern but diverges from mammalian gelatinase B in sequence and biochemical properties.

We have isolated a novel 75-kDa gelatinase from a chicken macrophage cell line, HD11. Biochemical and immunological characterization of the purified enzyme demonstrated that it is distinct from the chicken 72-kDa gelatinase A (MMP-2). The enzyme is capable of specific gelatin binding and rapid gelatin cleavage. Incubation with an organomercurial compound (p-aminophenylmercuric acetate) induces proteolytic processing and activation of this enzyme, and the resultant gelatinolytic activity is sensitive to both zinc chelators and tissue inhibitors of metalloproteinases. A full-length cDNA for the enzyme has been cloned, and sequence analysis demonstrated that the enzyme possesses the characteristic multidomain structure of an MMP gelatinase including a cysteine switch prodomain, three fibronectin type II repeats, a catalytic zinc binding region, and a hemopexin-like domain. The 75-kDa gelatinase is produced by phorbol ester-treated chicken bone marrow cells, monocytes, and polymorphonuclear leukocytes, cell types that charac- teristically produce the 92-kDa mammalian gelatinase B (MMP-9). The absence of a 90-110-kDa gelatinase in these cell types indicates that the 75-kDa gelatinase is likely the avian counterpart of gelatinase B. However, the protein is only 59% identical to human gelatinase B, whereas all previously cloned chicken MMP homologues are 75-90% identical to their human counterparts. In addition, the new 75-kDa chicken gelatinase lacks the type V collagen domain that is found in all mammalian gelatinase Bs. Furthermore, the secreted enzyme appears structurally distinct from known gelatinase Bs and the activated enzyme can cleave fibronectin, which is not a substrate for mammalian gelatinase B. Thus the results of this study indicate that a second MMP gelatinase exists in chickens, and although it is MMP-9/gelatinase B-like in its overall domain structure and expression pattern, it appears to be biochemically divergent from mammalian gelatinase B.

Amino Acid Sequence↗

Divergent cyclin B1 expression and Rb/p16/cyclin D1 pathway aberrations among pulmonary neuroendocrine tumors.

A total of 111 pulmonary neuroendocrine tumors comprising 13 typical carcinoids, five atypical carcinoids, 44 large-cell neuroendocrine carcinomas and 49 small-cell carcinomas were immunohistochemically studied for dysregulated cyclin B1 expression and disruption of the Rb/p16/cyclin D1 pathway (Rb pathway), and the results were correlated with tumor proliferation activity and clinical outcome. Overexpression of cyclins B1 and D1, respectively, was detected in no and 15% typical carcinoids, 20 and 20% atypical carcinoids, 84 and 32% large-cell neuroendocrine carcinomas, 84 and 10% small-cell carcinomas. Loss of Rb and p16 expression, respectively, was observed in no and 14% typical carcinoids, no and 40% atypical carcinoids, 49 and 18% large-cell neuroendocrine carcinomas, 84 and 8% small-cell carcinomas. In summary, 29% typical carcinoids, 20% atypical carcinoids, 78% large-cell neuroendocrine carcinomas and 93% small-cell carcinomas had Rb pathway aberrations. Rb pathway aberration was mostly attributed to Rb loss in small-cell carcinomas, while p16 loss and/or cyclin D1 overexpression besides Rb loss also played an important role in large-cell neuroendocrine carcinomas, while cyclin D1 overexpression was the only cause of Rb pathway aberration in carcinoid tumors. Thus, both cyclin B1-associated G2/M arrest and Rb-mediated G1 arrest are consistently compromised in high-grade large-cell neuroendocrine carcinoma and small-cell carcinoma, but are generally intact or occasionally altered in carcinoid tumor; the mechanisms involved in Rb pathway aberration among the tumor categories are different, reflecting a genetic divergence among the individual tumor categories. Cyclin B1 expression closely correlated with the Ki-67 labeling index either in the individual tumor categories or overall tumors (P < 0.0001, r = 0.742), suggesting that cyclin B1 is one of the key factors regulating cell proliferation in pulmonary neuroendocrine tumors. Neither cyclins B1 and D1, Rb, p16, nor Ki-67 correlated with patient survival in individual tumor categories, suggesting that the prognostic significance of these factors is tumor-type specific.

Biomarkers, Tumor↗

Gene loss, protein sequence divergence, gene dispensability, expression level, and interactivity are correlated in eukaryotic evolution.

Lineage-specific gene loss, to a large extent, accounts for the differences in gene repertoires between genomes, particularly among eukaryotes. We derived a parsimonious scenario of gene losses for eukaryotic orthologous groups (KOGs) from seven complete eukaryotic genomes. The scenario involves substantial gene loss in fungi, nematodes, and insects. Based on this evolutionary scenario and estimates of the divergence times between major eukaryotic phyla, we introduce a numerical measure, the propensity for gene loss (PGL). We explore the connection among the propensity of a gene to be lost in evolution (PGL value), protein sequence divergence, the effect of gene knockout on fitness, the number of protein-protein interactions, and expression level for the genes in KOGs. Significant correlations between PGL and each of these variables were detected. Genes that have a lower propensity to be lost in eukaryotic evolution accumulate fewer substitutions in their protein sequences and tend to be essential for the organism viability, tend to be highly expressed, and have many interaction partners. The dependence between PGL and gene dispensability and interactivity is much stronger than that for sequence evolution rate. Thus, propensity of a gene to be lost during evolution seems to be a direct reflection of its biological importance.

Amino Acid Substitution↗

Clustering of MAL genes in Hansenula polymorpha: cloning of the maltose permease gene and expression from the divergent intergenic region between the maltose permease and maltase genes.

Hansenula polymorpha uses maltase to grow on maltose and sucrose. Inspection of genomic clones of H. polymorpha showed that the maltase gene HPMAL1 is clustered with genes corresponding to Saccharomyces cerevisiae maltose permeases and MAL activator genes orthologues. We sequenced the H. polymorpha maltose permease gene HPMAL2 of the cluster. The protein (582 amino acids) deduced from the HPMAL2 gene is predicted to have eleven transmembrane domains and shows 39-57% identity with yeast maltose permeases. The identity of the protein is highest with maltose permeases of Debaryomyces hansenii and Candida albicans. Expression of the HPMAL2 in a S. cerevisiae maltose permease-negative mutant CMY1050 proved functionality of the permease protein encoded by the gene. HPMAL1 and HPMAL2 genes are divergently positioned similarly to maltase and maltose permease genes in many yeasts. A two-reporter assay of the expression from the HPMAL1-HPMAL2 intergenic region showed that expression of both genes is coordinately regulated, repressed by glucose, induced by maltose, and that basal expression is higher in the direction of the permease gene.

Amino Acid Sequence↗

Expression of the divergent tricarboxylate transport operon (tctI) of Salmonella typhimurium.

Membrane-associated gene products of shock-sensitive bacterial transport operons are often difficult to detect. A 4.5-kilobase DNA fragment, known to completely encode the Salmonella typhimurium tctI operon, was cloned in both orientations behind the T7 phage promoter phi 10 and expressed by using the T7 polymerase-promoter system of Tabor and Richardson (S. Tabor and C. C. Richardson, Proc. Natl. Acad. Sci. USA 82:1074-1078, 1985). Under these conditions, five proteins were clearly demonstrated. One DNA strand was shown to encode the periplasmic (29,000-Mr) C protein (as a 31,000-Mr precursor), a 19,000-Mr protein, and a 40,000- to 45,000-Mr protein which ran as a diffuse band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The opposite strand carried the information for two additional proteins of 29,000 and 14,000 Mr. By Tn5 mutagenesis, subcloning of Tn5 insertions, and subcloning of various deletion mutants it was shown that the tctI system is divergently transcribed. The periplasmic binding protein (C protein) is the first product of one operon, followed by the 19,000-Mr and 45,000-Mr integral inner membrane proteins. On the opposite strand only the 29,000-Mr protein was essential for tctI function, and it was found to be weakly attached to the inner membrane. Thus tctI encodes four proteins, one periplasmic, two integral, and one peripheral to the cytoplasmic membrane, with the genes arranged as tctA tctB tctC tctD.

Biological Transport↗

Effects of the su(Hw) insulator protein on the expression of the divergently transcribed Drosophila yolk protein genes.

The suppressor of Hairy-wing [su(Hw)] protein mediates the mutagenic effects of the gypsy retrotransposon by blocking enhancer activity. These repressive effects are general, can occur over long distances and require that the su(Hw) protein is bound between the affected enhancer and promoter. The effects of the su(Hw) binding region on yolk protein (yp) gene expression were determined. These genes are regulated by shared enhancers in the intergenic region, which provided a method to examine whether an enhancer blocked by the su(Hw) protein remained functional. We demonstrate that a blocked enhancer is completely active, supporting the proposal that the su(Hw) protein is an insulator protein that acts by forming a new boundary in a pre-existing chromatin domain, thereby preventing the interaction of regulatory elements located upstream of the insertion site with the promoter. In addition, we found that yp promoter function is not diminished by sharing enhancers, suggesting that these enhancers are not rate limiting for transcriptional activation. Lastly, our data indicate that yp promoter activity is interdependent, such that transcription from one promoter influences the level of activity of the linked promoter.

Alcohol Dehydrogenase↗

Seasonal plasticity of brain aromatase mRNA expression in glia: divergence across sex and vocal phenotypes.

Although teleost fishes have the highest levels of brain aromatase (estrogen synthase) compared to other vertebrates, little is known of its regulation and function in specific brain areas. Previously, we characterized the distribution of aromatase in the brain of midshipman fish, a model system for identifying the neural and endocrine basis of vocal-acoustic communication and alternative male reproductive tactics. Here, we quantified seasonal changes in brain aromatase mRNA expression in the inter- and intrasexually dimorphic sonic motor nucleus (SMN) and in the preoptic area (POA) in males and females in relation to seasonal changes in circulating steroid hormone levels and reproductive behaviors. Aromatase mRNA expression was compared within each sex throughout non-reproductive, pre-nesting, and nesting periods as well as between sexes within each season. Intrasexual (male) differences were also compared within the nesting period. Females had higher mRNA levels in the pre-nesting period when their steroid levels peaked, while acoustically courting (type I) males had highest expression during the nesting period when their steroid levels peaked. Females had significantly higher levels of expression than type I males in all brain areas, but only during the pre-nesting period. During the nesting period, non-courting type II males had significantly higher levels of aromatase mRNA in the SMN but equivalent levels in the POA compared to type I males and females. These results demonstrate seasonal and sex differences in brain aromatase mRNA expression in a teleost fish and suggest a role for aromatase in the expression of vocal-acoustic and alternative male reproductive phenotypes.

Analysis of Variance↗

Evaluation of hypothalamic gene expression in mice divergently selected for heat loss.

Mouse lines divergently selected for heat loss were evaluated for correlated responses in the hypothalamic transcriptome. High (MH) heat loss mice have approximately 50% greater heat loss, approximately 35% less body fat, approximately 20% greater feed intake, approximately 100% greater locomotor activity levels, and higher core body temperature compared with low (ML) heat loss mice. We evaluated hypothalamic expression between inbred lines derived from MH and ML lines (IH and IL, respectively) using cDNA microarrays and selected genes previously isolated in a large differential-display PCR experiment. Northern analysis was used to confirm differences, revealing higher hypothalamic mRNA expression of oxytocin (Oxt) and tissue inhibitor of metalloproteinase 2 (Timp-2) in the IH line. Real-time PCR assays were developed for Oxt, Timp-2, and ribosomal protein L3 (Rpl3, previously found to be upregulated in IL) and confirmed differential expression of these genes with potential physiological relevance in energy balance. These results provide information on correlated responses in the transcriptome of mice selected for high and low energy expenditure and reveal new information regarding genetic regulation of energy balance.

Animals↗

Organ-specific expression of highly divergent thionin variants that are distinct from the seed-specific crambin in the crucifer Crambe abyssinica.

Most thionins of higher plants are toxic to various bacteria, fungi, and animal and plant cells. The only known exception is the seed-specific thionin, crambin, of the crucifer Crambe abyssinica. Crambin has no net charge, is very hydrophobic and exhibits no toxicity. In the present work, the organization of the crambin precursor polypeptide was deduced from cD-NA sequences. The precursor shows a domain structure similar to that of the preproprotein of other thionins, which contains a signal peptide, a thionin domain and a C-terminal amino acid extension. Unlike the thionin precursors studied thus far, both the thionin domain and the C-terminal amino acid extension of the crambin precursor have no net charge and are hydrophobic, thus facilitating their interaction, by analogy to that proposed for the corresponding domains of other thionin precursors that have positive and negative charges. The existence of a large number of novel and highly variable thionin variants in Crambe abyssinica has been deduced from cDNA sequences that were amplified by the polymerase chain reaction (PCR) from RNA of seeds, leaves and cotyledons. While the deduced amino acid sequences of the thionin domains of most of these thionin precursor molecules are highly divergent, the two other domains are conserved. Most of the predicted thionin variants are positively charged. The presence of positively charged residues in the thionin domains consistently correlates with the presence of a negatively charged residue in the C-terminal amino acid extension of the various thionin precursors. The different thionin variants are encoded by distinct sets of genes and are expressed in an organ-specific manner.

Amino Acid Sequence↗

Expression of a divergent expansin gene is fruit-specific and ripening-regulated.

Expansins are proteins that induce extension in isolated plant cell walls in vitro and have been proposed to disrupt noncovalent interactions between hemicellulose and cellulose microfibrils. Because the plant primary cell wall acts as a constraint to cell enlargement, this process may be integral to plant cell expansion, and studies of expansins have focused on their role in growth. We report the identification of an expansin (LeExp1) from tomato that exhibits high levels of mRNA abundance and is specifically expressed in ripening fruit, a developmental period when growth has ceased but when selective disassembly of cell wall components is pronounced. cDNAs closely related to LeExp1 were also identified in ripening melons and strawberries, suggesting that they are a common feature of fruit undergoing rapid softening. Furthermore, the sequence of LeExp1 and its homologs from other ripening fruit define a subclass of expansin genes. Expression of LeExp1 is regulated by ethylene, a hormone known to coordinate and induce ripening in many species. LeExp1 is differentially expressed in the ripening-impaired tomato mutants Nr, rin, and nor, and mRNA abundance appears to be influenced directly by ethylene and by a developmentally modulated transduction pathway. The identification of a ripening-regulated expansin gene in tomato and other fruit suggests that, in addition to their role in facilitating the expansion of plant cells, expansins may also contribute to cell wall disassembly in nongrowing tissues, possibly by enhancing the accessibility of noncovalently bound polymers to endogenous enzymic action.

Amino Acid Sequence↗

A twin-reporter vector for simultaneous analysis of expression signals of divergently transcribed, contiguous genes in filamentous fungi.

To analyze the promoter region(s) of divergently transcribed fungal genes, a twin-reporter vector was constructed. This vector contains two divergently oriented reported genes, encoding Escherichia coli beta-glucuronidase (uidA) and E. coli beta-galactosidase (lacZ). Terminator regions of the Aspergillus nidulans nitrate and nitrite reductase-encoding genes, niaD and niiA, respectively, have been cloned 3' to the reporter genes to ensure proficient transcription termination of the reporter genes. The reporter genes have been separated by a unique NotI restriction site, which can be used for the insertion of expression signals. A mutant argB selection marker has been introduced in order to obtain A. nidulans transformants with a single copy of the vector integrated at the argB locus. The use of the vector was demonstrated by insertion of the A. nidulans niaD-niiA intergenic region and analysis of A. nidulans transformants obtained with this construct. Control of expression of both reporter genes was found to be in accordance with previously published data on control of nitrate assimilation [Cove, Biol. Rev. 54 (1979) 291-327].

Aspergillus nidulans↗

Regulation of expression of the divergent ulaG and ulaABCDEF operons involved in LaAscorbate dissimilation in Escherichia coli.

The ula regulon, responsible for the utilization of L-ascorbate in Escherichia coli, is formed by two divergently transcribed operons, ulaG and ulaABCDEF. The regulon is negatively regulated by a repressor of the DeoR family which is encoded by the constitutive gene ulaR located downstream of ulaG. Full repression of the ula regulon requires simultaneous interaction of the repressor with both divergent promoters and seems to be dependent on repressor-mediated DNA loop formation, which is helped by the action of integration host factor. Two operator sites have been identified in each promoter. Lack of either of the two sets of operators partially relieved the repression of the other operon; thus, each promoter is dependent on the UlaR operator sites of the other promoter to enhance repression. Electrophoretic mobility shift assays with purified UlaR protein and promoter deletion analyses revealed a conserved sequence, present in each of the four operators, acting as a UlaR binding site. Glucose represses the ula regulon via at least two mechanisms, one dependent on cyclic AMP (cAMP)-cAMP receptor protein (CRP) and the other (possibly inducer exclusion) independent of it. Glucose effects mediated by other global regulators cannot be ruled out with the present information. Changes in cAMP-CRP levels affected only the expression of the ulaABCDEF operon.

Ascorbic Acid↗

Two zebrafish eIF4E family members are differentially expressed and functionally divergent.

Eukaryotic translation initiation factor 4E (eIF4E) is an essential component of the translational machinery that binds m(7)GTP and mediates the recruitment of capped mRNAs by the small ribosomal subunit. Recently, a number of proteins with homology to eIF4E have been reported in plants, invertebrates, and mammals. Together with the prototypical translation factor, these constitute a new family of structurally related proteins. To distinguish the prototypical translation factor eIF4E from other family members, it has been termed eIF4E-1 (Keiper, B. D., Lamphear, B. J., Deshpande, A. M., Jankowska-Anyszka, M., Aamodt, E. J., Blumenthal, T., and Rhoads, R. E. (2000) J. Biol. Chem. 275, 10590-10596). We describe the characterization of two eIF4E family members in the zebrafish Danio rerio. Based on their relative identities with human eIF4E-1, these zebrafish proteins are termed eIF4E-1A (82%) and eIF4E-1B (66%). eIF4E-1B, originally termed eIF4E(L), has been reported previously as the zebrafish eIF4E-1 counterpart (Fahrenkrug, S. C., Dahlquist, M. O., Clark, K., and Hackett, P. B. (1999) Differentiation 65, 191-201; Fahrenkrug, S. C., Joshi, B., Hackett, P. B., and Jagus, R. (2000) Differentiation 66, 15-22). Sequence comparisons suggest that the two genes probably evolved from a duplication event that occurred during vertebrate evolution. eIF4E-1A is expressed ubiquitously in zebrafish, whereas expression of eIF4E-1B is restricted to early embryonic development and to gonads and muscle of the tissues investigated. The ability of these two zebrafish proteins to bind m(7)GTP, eIF4G, and 4E-BP, as well as to complement yeast conditionally deficient in functional eIF4E, show that eIF4E-1A is a functional equivalent of human eIF4E-1. Surprisingly, although eIF4E-1B possesses all known residues thought to be required for interaction with the cap structure, eIF4G, and 4E-BPs, it fails to interact with any of these components, suggesting that this protein serves a role other than that assigned to eIF4E.

Adaptor Proteins, Signal Transducing↗

Cloning and expression of a divergent integrin subunit beta 8.

Rabbit and human cDNA clones have been identified that encode a novel integrin beta subunit. The sequences that encode this subunit, which has been designated as beta 8, were isolated initially from rabbit placental cDNA libraries using an oligonucleotide probe derived from a highly conserved region of integrin beta subunit sequences. The rabbit clone was used to isolate human beta 8 cDNA clones from human placental and MG-63 osteosarcoma cell libraries. The putative beta 8 polypeptides, which comprise 769 and 768 residues in human and rabbit, respectively, show a high degree of inter-species conservation (approximately 90% identity). In contrast, beta 8 is distinct from the other integrin beta subunits. At the amino acid level human beta 8 ranges from 31 to 37% identity with human beta 1-7. The domain structure of beta 8 is typical of the integrin beta subunits. Human beta 8 has a 42-residue N-terminal signal peptide, a large extracellular domain (approximately 639 residues) that contains four cysteine-rich repeats, a transmembrane domain (approximately 30 residues), and a C-terminal cytoplasmic domain (approximately 58 residues). There are several structural features that are unique to the beta 8 polypeptide, as compared with the other integrin beta subunits. Six of the 56 cysteine residues that are conserved within the extracellular domains of beta 1, beta 2, beta 3, beta 5, beta 6, and the beta subunit from Drosophila are absent in the beta 8 polypeptide. Also, the cytoplasmic domain of the beta 8 subunit shares no homology with the cytoplasmic regions of any of the other integrin beta subunits. Northern analysis demonstrated an approximately 8-kilobase beta 8 mRNA in rabbit placenta, kidney, brain, ovary, and uterus. PCR analysis revealed that beta 8 mRNA is also present in several transformed human cell lines. The beta 8 polypeptide has been transiently expressed in 293 human embryonic kidney cells. A polyclonal antipeptide antibody specific for beta 8 and a polyclonal antibody that recognizes alpha v epitopes were used to show that beta 8 can complex with the endogenous alpha v subunit in 293 cells and that the resulting integrin is expressed as a cell surface complex.

Amino Acid Sequence↗

DNA bending and expression of the divergent nagE-B operons.

Repression of the divergent nagE - B operons requires NagC binding to two operators which overlap the nagE and nagB promoters, resulting in formation of a DNA loop. Binding of the cAMP/CAP activator to its site, adjacent to the nagE operator, stabilizes the DNA loop in vitro. The DNA of the nagE-B intergenic region is intrinsically bent, with the bend centred on the CAP site. We show that displacement of the CAP site by 6 bp results in complete derepression of the two operons. This derepression is observed even in the absence of cAMP/CAP binding and despite the fact that the two NagC operators are still in phase, demonstrating that the inherently bent structure of the DNA loop is important for repression. Since no interaction between NagC and CAP has been detected, we propose that the role of CAP in the repression loop is architectural, stabilizing the intrinsic bend. The cAMP/CAP complex is necessary for activation of the nagE-B promoters. In this case protein-protein contacts between CAP and RNA polymerase are necessary for full activation, but at least a part of the activation is likely due to an effect of CAP binding altering DNA structure.

Acetylglucosamine↗

Divergence of gene expression in neurohypophysial hormone precursors among salmonids.

Salmonid fish have pairs of genes for various hypothalamic and pituitary hormones including neurohypophysial hormones, vasotocin, and isotocin, probably because they are tetraploid. The problem here is whether two genes for the same hormone are expressed equally or differently. We therefore examined expression patterns of vasotocin and isotocin genes in four salmonid species using Northern blot analysis with chum salmon cDNAs as hybridization probes. The presence of two vasotocin and also two isotocin genes was confirmed by Southern blot analysis in rainbow trout and sockeye salmon which were not examined previously. Prior to Northern blot analysis, isotocin-I cDNA of sockeye salmon was determined and compared to those of chum salmon and masu salmon, since molecular probes are so specific that cross-species hybridization often leads misinterpretation in a quantitative study. The nucleotide sequence of sockeye salmon isotocin-I cDNA showed sufficiently high homology (> 96.0%) to those of chum salmon and masu salmon for cross-species hybridization among salmonids. Northern blot analysis showed that both isotocin-I and isotocin-II genes were well expressed in all species examined. Expression of isotocin-I gene tended to be relatively higher than that of isotocin-II gene in all species. However, expression pattern of vasotocin-I and vasotocin-II genes did not coincide among species. Expression of vasotocin-II genes was very weak or scarce in masu salmon and rainbow trout, while that in sockeye salmon was stronger than vasotocin-I gene expression. The present result may reflect complicated molecular evolution of salmonid vasotocin genes probably both in regulatory and coding regions.

Amino Acid Sequence↗

Overlapping divergent promoters control expression of Tn10 tetracycline resistance.

We have previously examined the genetic organization and regulation of the Tn10 tetracycline-resistance determinant in Escherichia coli K-12. The structural genes for tetA, the Tn10 tetracycline-resistance function, and for tetR, the Tn10 tet repressor, are transcribed in opposite directions from promoters in a regulatory region located between the two structural genes. Expression of both tetA and tetR is induced by tetracycline. Here we report the DNA sequence of the Tn10 tet regulatory region. The locations of the tetA and tetR promoters within this region were defined by S1 nuclease mapping of the 5' ends of in vivo tet RNA. The tetA and tetR promoters overlap; the transcription start points are separated by 36 bp. We propose that two similar regions of dyad symmetry within the Tn10 tet regulatory region are operator sites at which tet repressor binds to tet DNA, thereby inhibiting transcription initiation at the tetA and tetR promoters. The Tn10 tet regulatory region and the pBR322 tet regulatory region show significant DNA sequence homology (53%).

Base Sequence↗