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PubMed1998

Use of asymmetric cell division and spoIIIE mutants to probe chromosome orientation and organization in Bacillus subtilis.

Soon after the onset of sporulation in Bacillus subtilis, asymmetric cell division occurs to generate the differentiating prespore and mother cell types. Formation of the septum close to the cell pole initially bisects the nucleoid destined for the prespore, trapping only about one-third of the DNA in the small compartment. The remaining part of the chromosome is then transported through the septum. spoIIIE mutant cells fail to transfer the DNA and arrest with only partially segregated prespore chromosomes. Previous work has shown that the orientation of the chromosome at the time of septation is not random. Here, we use both physical and genetic methods to characterize the trapped DNA. The results show that the chromosome has a very specific orientation at the time of septation, consistent with the action of a centromere-like sequence near oriC. They also demonstrate that the chromosome is folded, or otherwise organized, in a highly ordered manner.

Bacillus subtilis↗

PubMed2000

Novel features of the XRN-family in Arabidopsis: evidence that AtXRN4, one of several orthologs of nuclear Xrn2p/Rat1p, functions in the cytoplasm.

The 5'-3' exoribonucleases Xrn1p and Xrn2p/Rat1p function in the degradation and processing of several classes of RNA in Saccharomyces cerevisiae. Xrn1p is the main enzyme catalyzing cytoplasmic mRNA degradation in multiple decay pathways, whereas Xrn2p/Rat1p functions in the processing of rRNAs and small nucleolar RNAs (snoRNAs) in the nucleus. Much less is known about the XRN-like proteins of multicellular eukaryotes; however, differences in their activities could explain differences in mRNA degradation between multicellular and unicellular eukaryotes. One such difference is the lack in plants and animals of mRNA decay intermediates like those generated in yeast when Xrn1p is blocked by poly(G) tracts that are inserted within mRNAs. We investigated the XRN-family in Arabidopsis thaliana and found it to have several novel features. First, the Arabidopsis genome contains three XRN-like genes (AtXRNs) that are structurally similar to Xrn2p/Rat1p, a characteristic unique to plants. Furthermore, our experimental results and sequence database searches indicate that Xrn1p orthologs may be absent from higher plants. Second, the lack of poly(G) mRNA decay intermediates in plants cannot be explained by the activity of the AtXRNs, because they are blocked by poly(G) tracts. Finally, complementation of yeast mutants and localization studies indicate that two of the AtXRNs likely function in the nucleus, whereas the third acts in the cytoplasm. Thus, the XRN-family in plants is more complex than in other eukaryotes, and, if an XRN-like enzyme plays a role in mRNA decay in plants, the likely participant is a cytoplasmic Xrn2p/Rat1p ortholog, rather than an Xrn1p ortholog.

Amino Acid Sequence↗

PubMed1997

Molecular evolution at the decapentaplegic locus in Drosophila.

Using an elaborate set of cis-regulatory sequences, the decapentaplegic (dpp) gene displays a dynamic pattern of gene expression during development. The C-terminal portion of the DPP protein is processed to generate a secreted signaling molecule belonging to the transforming growth factor-beta (TGF-beta) family. This signal, the DPP ligand, is able to influence the developmental fates of responsive cells in a concentration-dependent fashion. Here we examine the sequence level organization of a significant portion of the dpp locus in Drosophila melanogaster and use interspecific comparisons with D. simulans, D. pseudoobscura and D.virilis to explore the molecular evolution of the gene. Our interspecific analysis identified significant selective constraint on both the nucleotide and amino acid sequences. As expected, interspecific comparison of protein coding sequences shows that the C-terminal ligand region is highly conserved. However, the central portion of the protein is also conserved, while the N-terminal third is quite variable. Comparison of noncoding regions reveals significant stretches of nucleotide identity in the 3' untranslated portion of exon 3 and in the intron between exons 2 and 3. An examination of cDNA sequences representing five classes of dpp transcripts indicates that these transcripts encode the same polypeptide.

Amino Acid Sequence↗

PubMed1979

Isolation and characterization of cloned human fetal globin genes.

Three clones containing both the human G gamma and A gamma globlin genes have been isolated and characterized from a library of DNA fragments generated by partial Eco RI digestion of cellular DNA using charon 4A phage as vector. Two of the clones (NY 2 and 3) are identical and have an insert of 14.0 kb. The third clone (NY 1) has a 15.4 kb insert by virtue of an extra 1.4 kb Eco RI fragment at its 5' most end. This clone also has a Kpn I site not present in the other two suggesting it is the product of the gamma gene on the opposite chromosome. Restriction analysis of the three clones indicates that the G gamma and A gamma genes are linked on a single continuous piece of DNA and are separated by 3.5 kb and each contains at least one large intervening sequence of 0.85 kg between the Bam HI and Eco RI sites. These findings in cloned DNA provide direct evidence for linkage and organization of the gamma genes in man.

Base Sequence↗

PubMed1987

Comparative structure of the protease-sensitive regions of the subfragment-1 heavy chain from smooth and skeletal myosins.

The heavy chain fragments generated by restricted proteolysis of the smooth chicken gizzard myosin subfragment-1 (S-1) with trypsin, Staphylococcus aureus V8 protease, and chymotrypsin were isolated and submitted to partial amino acid sequencing. The comparison between the smooth and striated muscle myosin sequences permitted the unambiguous structural characterization of the two protease-vulnerable segments joining the three putative domain-like regions of the smooth head heavy chain. The smooth carboxyl-terminal connector is a serine-rich region located around positions 632-640 of the rabbit skeletal sequence and would represent the "A" site that is conformationally sensitive to the myosin 10 S-6 transition and to its interaction with actin (Ikebe, M., and Hartshorne, D. J. (1986) Biochemistry 25, 6177-6185). A third site which undergoes a nucleotide-dependent chymotryptic cleavage which inactivates the Mg2+-ATPase (Okamoto, Y., and Sekine, T. (1981) J. Biochem. (Tokyo) 90, 833-842, 843-849) was identified at Trp-31/Ser-32. It is vicinal to Lys-34 that is monomethylated in the skeletal heavy chain but not at all in the smooth sequence. However, the two trimethyl lysine residues present in the skeletal sequence are conserved in the same regions of the smooth S-1 and may play a general functional role in myosin. The smooth central 50-kDa segment could be selectively destroyed by a mild tryptic digestion in the absence of any unfolding agent, with a concomitant inhibition of the ATPase activities. This feature is in line with the proposed domain structure of the S-1 heavy chain and also suggests a relationship between the specific biochemical properties of the smooth S-1 and the particular conformation of its 50-kDa region.

Amino Acid Sequence↗

PubMed1999

Random mutagenesis of the pomA gene encoding a putative channel component of the Na(+)-driven polar flagellar motor of Vibrio alginolyticus.

PomA and PomB are integral membrane proteins and are essential for the rotation of the Na(+)-driven polar flagellar motor of Vibrio alginolyticus. On the basis of their similarity to MotA and MotB, which are the proton-conducting components of the H(+)-driven motor, they are thought to form the Na(+)-channel complex and to be essential for mechanochemical coupling in the motor. To investigate PomA function, random mutagenesis of the pomA gene by using hydroxylamine was carried out. We isolated 37 non-motile mutants (26 independent mutations) and most of the mutations were dominant; these mutant alleles are able to inhibit the motility of wild-type cells when greatly overexpressed. The mutant PomA proteins could be detected by immunoblotting, except for those with deletions or truncations. Many of the dominant mutations were mapped to the putative third or fourth transmembrane segments, which are the most conserved regions. Some mutations that showed strong dominance were in highly conserved residues. T1861 is the mutation of a polar residue located in a transmembrane segment that might be involved in ion translocation. P199L occurred in a residue that is thought to mediate conformational changes essential for torque generation in MotA. These results suggest that PomA and MotA have very similar structures and roles, and the basic mechanism for torque generation will be similar in the proton and sodium motors.

Amino Acid Sequence↗

PubMed2000

A common polymorphism in the human insulin-like growth factor binding protein 3 gene.

A common polymorphism generated by an A to C transition at nucleotide 7580 in the third intron of the insulin-like growth factor binding protein 3 gene was found. This polymorphism can be identified by Nde 1 restriction of genomic DNA amplified with a specifically designed restriction enzyme site-generating oligonucleotide primer. The frequency of the A and C alleles was estimated at 0.6 and 0.4 in the Caucasians, 0.63 and 0.37 in Blacks, respectively.

Amino Acid Substitution↗

PubMed2002

Contribution of regions distal to glycine-160 to the anticoagulant activity of tissue factor pathway inhibitor.

The functions of the first two Kunitz domains of tissue factor pathway inhibitor (TFPI) are well defined as active site-directed inhibitors of factor VIIa and factor Xa. The anticoagulant properties of the third Kunitz domain and C-terminal region were probed using altered forms of TFPI. TFPI-160 contains the first two Kunitz domains. K1K2C contains the first two Kunitz domains and the basic C-terminus. Neither TFPI-160 nor K1K2C contains the third Kunitz domain. In amidolytic assays containing calcium, TFPI-160 is a less potent inhibitor of factor Xa than TFPI. However, addition of the C-terminus in K1K2C nearly restores inhibitory activity to that of TFPI, indicating that the third Kunitz domain is not required for direct inhibition of factor Xa. When compared in assays containing phospholipids and factor Va, K1K2C and TFPI-160 are poor inhibitors compared to TFPI, demonstrating that the third Kunitz domain is required for the full anticoagulant activity of TFPI. TFPI was further characterized in amidolytic assays performed with Gla-domainless factor Xa and in prothrombin activation assays using submicellar concentrations of short-chain phospholipids (C6PS). TFPI and K1K2C are worse inhibitors of Gla-domainless factor Xa, compared to wild-type factor Xa, while TFPI-160 inhibits both forms of factor Xa equally, suggesting a C-terminus/Gla domain interaction. TFPI is a potent inhibitor of thrombin generation by prothrombinase assembled with C6PS, while TFPI-160 and K1K2C are not. Conversely, TFPI does not inhibit prothrombin activation by prothrombinase assembled on a two-dimensional lipid bilayer. Together, the data indicate that the region between Gly-160 and the end of the third Kunitz domain contributes to TFPI function by orienting the second Kunitz domain so that it can bind the active site of phospholipid-associated factor Xa prior to prothrombinase assembly and/or by slowing formation of the prothrombinase complex.

Amino Acid Sequence↗

PubMed2000

The 21- and 23-kD forms of TCR zeta are generated by specific ITAM phosphorylations.

The T cell receptor (TCR) zeta subunit contains three immunoreceptor tyrosine-based activation motifs (ITAMs) that translate effective extracellular ligand binding into intracellular signals by becoming phosphorylated into 21- and 23-kD forms. We report here that the 21-kD form of TCR zeta is generated by phosphorylation of the tyrosines in the second and third ITAMs, whereas the 23-kD form is formed by the additional phosphorylation of the membrane-proximal ITAM tyrosines. The stable formation of the 21- and 23-kD species requires the binding of the tandem SH2 domains of ZAP-70. We also report that TCR-mediated signaling processes can proceed independently of either the 21- or 23-kD species of TCR zeta.

Amino Acid Sequence↗

PubMed1995

Retroviral-mediated gene transfer in human bone marrow cells growth in continuous perfusion culture vessels.

Hematopoietic stem cell gene therapy holds the promise of being able to treat a variety of inherited and acquired diseases of the hematopoietic stem cell. However, to date, genetic modification of the human hematopoietic stem cell has been relatively inefficient. Here, we report the results of using a bioreactor system to expand hematopoietic cells after a brief retrovirus infection using a high titer, replication defective virus encoding for murine CD18. The retrovirus transduced culture continued to produce genetically modified hematopoietic progenitors for up to 6 weeks, the duration of the culture period. Up to one-third of the long-term culture initiating cell (LTC-IC) are genetically modified by the culture conditions. Murine CD18 can be expressed on the cell surface of up to 20% of the mature cells generated by the culture system, suggesting that clinically significant levels of gene transfer may be occurring. These results demonstrate the feasibility of using continuous perfusion bioreactors as a method of efficiently modifying human hematopoietic stem cells.

Animals↗

PubMed1987

In-vivo degradation pathway of an excised intervening sequence.

A study of the distribution in heterogeneous nuclear RNP (hnRNP) of the different forms of the first intervening sequence (IVSI) from the E3 transcription unit of adenovirus serotype 2 suggests a three-step pathway for IVS excision and degradation. First, the lariat IVS is formed in a large RNP of the size of premessenger RNP where it is resistant to nuclease attack. Second, the lariat IVS is released from this large structure as a small RNP where it is very susceptible to enzymic degradation. Third, the lariat IVS is linearized after the release of the small RNP. Our studies suggest that only the intact lariat and linear IVS are involved in the excision-degradation pathway in vivo, whereas the lariat nicked in its circular part and the IVS trimmed at its 3' end are not pre-existing but generated during isolation of hnRNA or hnRNP.

Adenoviruses, Human↗

PubMed2003

A comparison of DNA vaccines for the rabies-related virus, Mokola.

Mokola virus, a rabies-related virus, has been reported to date from the African continent only. Like rabies virus, it is highly pathogenic, causes acute encephalitis, and zoonotic events have been documented. Although believed to be rare, there has been an unexplained increase in the number of isolations of the virus in South Africa in recent years. We have cloned and sequenced the glycoprotein (G) and nucleoprotein (N) genes from a South African Mokola virus, and used these in the construction of different DNA vaccines for immunization against Mokola virus. Four vaccines, utilizing different promoters and DNA backbone compositions, were generated and compared for efficacy in protection against Mokola virus. In one of these, both the Mokola virus G and N genes were co-expressed. Two of the single G-expressing DNA vaccines (based on pSG5 and pCI-neo, respectively) protected laboratory mice against lethal challenge, despite major differences in their promoters. However, neither vaccine was fully protective in a single immunization only. Serological assays confirmed titers of virus-neutralizing antibodies after immunization, which increased upon booster vaccine administration. A third construct (based on pBudCE4) was less effective in inducing a protective immune response, despite employing a strong CMV enhancer/promoter also used in the pCI-neo plasmid. Dual expression of Mokola virus G and N genes in pBudCE4 did not enhance its efficacy, under the conditions described. In addition, no significant utility could be demonstrated for a combined prime-boost approach, as no cross-protective immunity was observed against rabies or Mokola viruses from the use of pSG5-mokG or vaccinia-rabies glycoprotein recombinant virus vaccines, respectively, even though both vaccines provided 60-100% protection against homologous virus challenge.

Animals↗

PubMed1998

Characterization of aggrecan retained and lost from the extracellular matrix of articular cartilage. Involvement of carboxyl-terminal processing in the catabolism of aggrecan.

The catabolism of aggrecan in bovine articular cartilage explants is characterized by the release into the culture medium of high molecular weight aggrecan fragments, generated by the proteolytic cleavage of the core protein between residues Glu373 and Ala374 within the interglobular domain. In this study, the position of the carboxyl-terminus of these aggrecan fragments, as well as a major proteolytically shortened aggrecan core protein present in cartilage matrix, have been deduced by characterizing the peptides generated by the reaction of aggrecan core protein peptides with cyanogen bromide. It was shown that two out of three such peptide fragments having an amino terminus starting at Ala374 have their carboxyl terminus located within the chondroitin sulfate 1 domain. The third and largest aggrecan core protein peptide, with an amino terminus starting at Ala374, has a carboxyl terminus in a region of core protein between the chondroitin sulfate 1 domain and the chondroitin sulfate 2 domain. The carboxyl terminus of this peptide appeared to be the same as that of the proteolytically degraded aggrecan core protein, which is retained within the extracellular matrix of the tissue. Another two aggrecan fragments recovered from the medium of explant cultures with amino-terminal sequences in the chondroitin sulfate 2 domain at Ala1772 and Leu1872 were shown to have their carboxyl termini within the G3 globular domain. These results suggest that the catabolism of aggrecan between residues Glu373 and Ala374 in the interglobular domain by the putative proteinase, aggrecanase, may be dependent on prior proteolytic processing within the carboxyl-terminal region of the core protein.

Aggrecans↗

PubMed2006

Developmental constraint of insect audition.

BACKGROUND: Insect ears contain very different numbers of sensory cells, from only one sensory cell in some moths to thousands of sensory cells, e.g. in cicadas. These differences still await functional explanation and especially the large numbers in cicadas remain puzzling. Insects of the different orders have distinct developmental sequences for the generation of auditory organs. These sensory cells might have different functions depending on the developmental stages. Here we propose that constraints arising during development are also important for the design of insect ears and might influence cell numbers of the adults. PRESENTATION OF THE HYPOTHESIS: We propose that the functional requirements of the subadult stages determine the adult complement of sensory units in the auditory system of cicadas. The hypothetical larval sensory organ should function as a vibration receiver, representing a functional caenogenesis. TESTING THE HYPOTHESIS: Experiments at different levels have to be designed to test the hypothesis. Firstly, the neuroanatomy of the larval sense organ should be analyzed to detail. Secondly, the function should be unraveled neurophysiologically and behaviorally. Thirdly, the persistence of the sensory cells and the rebuilding of the sensory organ to the adult should be investigated. IMPLICATIONS OF THE HYPOTHESIS: Usually, the evolution of insect ears is viewed with respect to physiological and neuronal mechanisms of sound perception. This view should be extended to the development of sense organs. Functional requirements during postembryonic development may act as constraints for the evolution of adult organs, as exemplified with the auditory system of cicadas.

Journal Article↗

PubMed1992

Purification and characterization of Rac 2. A cytosolic GTP-binding protein that regulates human neutrophil NADPH oxidase.

Human neutrophils and other phagocytes generate superoxide anion (O2-) as a means of destroying ingested microorganisms. O2- is produced by an NADPH-consuming oxidase composed of membrane and cytosolic components. Activation of the NADPH oxidase is absolutely dependent upon GTP, indicating the requirement for a GTP-binding protein in this process. We have utilized a five-step chromatographic procedure to isolate a GTP-binding protein from human neutrophil cytosol which can stimulate NADPH oxidase activity in a cell-free assay. Oxidase enhancing activity was shown to coisolate with this GTP-binding component, which was purified to apparent homogeneity. The GTP-binding protein was identified as Rac 2 by immunological reactivity and amino acid sequencing. Thus, Rac 2 appears to be a third cytosolic component required for human neutrophil NADPH oxidase activation. Recombinant Rac 2 was shown to bind guanine nucleotides in a Mg(2+)-dependent fashion. GDP dissociation rates were determined and shown to be regulated by the free Mg2+ concentration. Rac 2 was found to possess the highest rate of intrinsic GTP hydrolysis of any of the characterized members of the Ras superfamily. The biochemical properties of Rac 2 indicate it is likely to be subject to regulatory cofactors in vivo.

Chromatography, Affinity↗

PubMed1996

Analysis of the structure and expression of the augmenter of liver regeneration (ALR) gene.

BACKGROUND: The gene encoding the hepatotrophic factor Augmenter of Liver Regeneration (ALR) has recently been cloned in the rat. The availability of the mouse form of ALR would allow the analysis of the role of this factor in the physiology of liver and other organs, while the identification of the human homolog would allow the transfer of the great wealth of information that has been generated in animal models to clinically oriented pilot trials, and eventually the therapeutic application of this information. MATERIALS AND METHODS: Standard molecular biology approaches have been used to determine the genomic structure of the ALR gene in the mouse, and to characterize the ALR transcript and its protein product. The human ALR cDNA was also isolated and the amino acid sequence of the human gene product deduced. The mapping of mouse and human ALR genes on mouse and human chromosomes was then completed. RESULTS: The protein coding portion of the mouse ALR gene is comprised of three exons, the first containing the 5' untranslated sequence and the initial 18 bases after the ATG translation initiation codon, the second exon encompasses 198 bases, and the third exon contains the remaining portion of the protein coding sequence. Rat, mouse, and human ALR genes (and protein products) were found to be highly conserved and preferentially expressed in the testis and in the liver. The ALR gene maps to the mouse chromosome 17, in a region syntenic with human chromosome 16, where the T/t region has also been mapped. CONCLUSIONS: ALR appears to be a protein with important physiologic properties, not exclusively limited to liver regeneration, with roles that are involved in the synthesis or stability of the nuclear and mitochondrial transcripts that are present in actively regenerating cells, particularly the germ cells of the testes.

Amino Acid Sequence↗

PubMed1998

Characterization of helical cleavages in type II collagen generated by matrixins.

Several vertebrate collagenases have been reported to cleave type II collagen, leading to irreversible tissue destruction in osteoarthritis. We have investigated the action of MMP-1 and MMP-13 on type II collagen by use of neoepitope antibodies and N-terminal sequencing. Previous studies have suggested that the initial cleavage of type II collagen by MMP-13 is followed by a second cleavage, three amino acids carboxy-terminal to the primary cleavage site. We show here that this cleavage is also produced by APMA-activated MMP-1 in combination with MMP-3 (i.e. fully activated MMP-1). The use of a selective inhibitor of MMP-3 has shown that it is this enzyme, rather than interstitial collagenase which had been exposed to MMP-3, which makes the second cleavage. In addition we have identified, through N-terminal sequencing, a third cleavage site, three residues carboxy-terminal to the secondary site. Since MMP-2 is thought to be responsible for gelatinolytic action on type II collagen we have investigated the effect of MMP-2 after the initial helical cleavage made by either MMP-1 or MMP-13. A combination of MMPs-1, -2 and -3 results in both the second and third cleavage sites; adding MMP-2 to MMP-13 did not alter the cleavage pattern produced by MMP-13 on its own. We conclude that none of the three cleavage sites will provide information about the specific identity of the collagenolytic enzymes involved in collagen cleavage in situ. Staining of cartilage sections of osteoarthritis patients with the neoepitope antibodies revealed type II collagen degradation starting at or near the articular surface and extending into the mid and deep zones with increasing degeneration of the cartilage.

Animals↗

PubMed1995

Hyperthermostable mutants of Bacillus licheniformis alpha-amylase: multiple amino acid replacements and molecular modelling.

We have identified previously two critical positions for the thermostability of the highly thermostable alpha-amylase from Bacillus licheniformis. We have now introduced all 19 possible amino acid residues to these two positions, His133 and Ala209. The most favourable substitutions were to Ile and Val, respectively, which both increased the half-life of the enzyme at 80 degrees C by a factor of approximately 3. At both positions a stabilizing effect of hydrophobic residues was observed, although only in the case of position 133 could a clear correlation be drawn between the hydrophobicity of the inserted amino acid and the gain in protein stability. The construction of double mutants showed a cumulative effect of the most favourable and/or deleterious substitutions. Computer modelling was used to generate a 3-D structure of the wild-type protein and to model substitutions at position 209, which lies in the conserved (alpha/beta)8 barrel domain of alpha-amylase; Ala209 would be located at the beginning of the third helix of the barrel, in the bottom of a small cavity facing the fourth helix. The model suggests that replacement by, for example, a valine could fill this cavity and therefore increase intra- and interhelical compactness and hydrophobic interactions.

Amino Acid Sequence↗