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PubMed1997

Functional analysis of in vivo and in organello footprinting of HeLa cell mitochondrial DNA in relationship to ATP and ethidium bromide effects on transcription.

In vivo and in organello footprinting techniques based on methylation interference have been utilized to investigate protein-DNA interactions in the transcription initiation and rDNA transcription termination regions of human mitochondrial DNA (mtDNA) in functionally active mitochondria. In particular, the changes in methylation reactivity of these regions in response to treatment of the organelles with ATP or ethidium bromide, which affects differentially the rates of mitochondrial rRNA and mRNA synthesis, have been analyzed. Two major sites of protein-DNA interactions have been identified in the main control region of mtDNA, both in vivo and in organello, which correspond to the regions of the light-strand promoter and heavy-strand rRNA-specific promoter. The in organello footprinting of the latter showed ATP- and ethidium bromide-dependent modifications that could be correlated with changes in the rate of rRNA but not of mRNA synthesis. By contrast, no ATP effects were observed on the in organello footprinting pattern of the termination region and on in vitro transcription termination, strongly suggesting that ATP control of rRNA synthesis occurs at the initiation level. Several methylation interference sites were found upstream of the whole H-strand transcription unit, pointing to possible protein-DNA interactions related to the activity of this unit. In vivo footprinting of the rDNA transcription termination region of human mtDNA has revealed a very strong protection pattern, indicating a high degree of occupancy of the termination site by mitochondrial transcription termination factor (approximately 80%).

Adenosine Triphosphate↗
PubMed2002

Functional analysis of cdc42 residues required for Guanine nucleotide exchange.

Guanine nucleotide exchange factors (GEFs) directly engage small GTPases to facilitate the exchange of bound GDP for GTP, leading to GTPase activation. Several recent crystal structures of GEFs in complex with Rho family GTPases highlight the conserved interactions and conformational alterations necessary for catalyzing exchange. In the present study, functional roles were defined for specific residues within Cdc42 implicated by the crystal structures as important for physiological exchange of guanine nucleotides within Rho GTPases. In particular, this study highlights the paramount importance of the phosphate-binding loop and interactions with the magnesium co-factor as critical for proper regulation of RhoGEF-catalyzed exchange. Other conformational alterations of the GTPases affecting interactions with the sugar and base of guanine nucleotides are also important but are secondary. Of particular note, substitution of alanine for cysteine at position 18 of Cdc42 leads to a fast cycling phenotype for Cdc42 with heightened affinity for RhoGEFs and produces a dominant negative form of Cdc42 capable of inhibiting RhoGEFs both in vitro and in vivo.

Cloning, Molecular↗
PubMed1997

Functional analysis of the Shigella flexneri IpaC invasin by insertional mutagenesis.

The ability of Shigella to enter epithelial cells, to escape from the phagocytic vacuole, and to induce apoptosis in macrophages requires the IpaB, IpaC, and IpaD proteins. An extracellular complex containing IpaB and IpaC can promote the uptake of inert particles by epithelial cells. To determine whether the function of IpaC is to act as an extracellular chaperone for IpaB in the Ipa complex or as an effector of entry involved in a direct interaction with the cell surface, we have constructed eight IpaC recombinant proteins by inserting the coding sequence for a 12- to 14-amino-acid fragment into restriction sites scattered within the ipaC gene. We have investigated the ability of recombinant proteins to bind IpgC in the bacterial cytoplasm and IpaB in the extracellular medium and to complement an ipaC null mutant for entry into HeLa cells, lysis of erythrocytes, and escape from the phagocytic vacuole in infected macrophages. Most recombinant proteins were produced and secreted at a level similar to that of wild-type IpaC and did not exhibit altered susceptibility to proteolysis by trypsin, and all were able to bind IpgC and IpaB. Some recombinant proteins did not complement the ipaC mutant for entry into HeLa cells, lysis of erythrocytes, or escape from the phagocytic vacuole, which indicates that IpaC plays an active role in these processes and does not act solely as a chaperone for IpaB. In addition, some insertions which were located outside of the hydrophobic region of IpaC differentially affected the abilities of Shigella to enter epithelial cells and to lyse cell membranes.

Antibodies, Monoclonal↗
PubMed1996

Functional analysis of the mutations in the human cardiac beta-myosin that are responsible for familial hypertrophic cardiomyopathy. Implication for the clinical outcome.

More than 30 missense mutations in the beta-cardiac myosin heavy chain gene have been shown to be responsible for familial hypertrophic cardiomyopathy. To clarify the effects of these point mutations on myosin motor function, we expressed wild-type and mutant human beta-cardiac myosin heavy chains in insect cells with human cardiac light chains. The wild-type myosin was well purified with similar enzymatic and motor activities to those of the naturally isolated V3 cardiac myosin. Arg249-->Gln and Arg453-->Cys mutations resulted in decreased actin translocating activity (61 and 23% of the wild-type, respectively) with decreased intrinsic ATPase activity. Arg403-->Gln mutation greatly decreased actin translocating activity (27% of wild type) with a 3.3-fold increased dissociation constant for actin, while intrinsic ATPase activity was unchanged. Val606-->Met mutation only mildly affected the actin translocating activity as well as ATPase activity of myosin. The degree of deterioration by each mutation was closely correlated with the prognosis of the affected kindreds, indicating that myosin dysfunction caused by the point mutations is responsible for the pathogenesis of the disease. Structure/function relationship of myosin is discussed.

Actins↗
PubMed2003

Functional analysis of the antimitogenic activity of tumor suppressors.

Loss of tumor suppressors contributes to numerous cancer types. Many, but not all, proteins encoded by tumor suppressor genes have antiproliferative activity and halt cell-cycle progression. In this chapter, we present three methods that have been utilized to monitor the antimitogenic action exerted by tumor suppressors. Tumor suppressor function can be demonstrated by colony formation assays and acquisition of the flat-cell phenotype. Because of the anti-proliferative action of these agents, we also present two transient assays that monitor the effect of tumor suppressors on cell-cycle progression. One is based on BrdU incorporation (i.e., DNA replication) and the other on flow cytometry. Together, this triad of techniques is sufficient to determine the action of tumor suppressors and other antiproliferative agents.

Antimetabolites↗
PubMed1999

Molecular cloning and functional analysis of SUT-1, a sulfate transporter from human high endothelial venules.

High endothelial venules (HEV) are specialized postcapillary venules found in lymphoid organs and chronically inflamed tissues that support high levels of lymphocyte extravasation from the blood. One of the major characteristics of HEV endothelial cells (HEVEC) is their capacity to incorporate large amounts of sulfate into sialomucin-type counter-receptors for the lymphocyte homing receptor L-selectin. Here, we show that HEVEC express two functional classes of sulfate transporters defined by their differential sensitivity to the anion-exchanger inhibitor 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS), and we report the molecular characterization of a DIDS-resistant sulfate transporter from human HEVEC, designated SUT-1. SUT-1 belongs to the family of Na(+)-coupled anion transporters and exhibits 40-50% amino acid identity with the rat renal Na(+)/sulfate cotransporter, NaSi-1, as well as with the human and rat Na(+)/dicarboxylate cotransporters, NaDC-1/SDCT1 and NaDC-3/SDCT2. Functional expression studies in cRNA-injected Xenopus laevis oocytes showed that SUT-1 mediates high levels of Na(+)-dependent sulfate transport, which is resistant to DIDS inhibition. The SUT-1 gene mapped to human chromosome 7q33. Northern blotting analysis revealed that SUT-1 exhibits a highly restricted tissue distribution, with abundant expression in placenta. Reverse transcription-PCR analysis indicated that SUT-1 and the diastrophic dysplasia sulfate transporter (DTD), one of the two known human DIDS-sensitive sulfate transporters, are coexpressed in HEVEC. SUT-1 and DTD could correspond, respectively, to the DIDS-resistant and DIDS-sensitive components of sulfate uptake in HEVEC. Together, these results demonstrate that SUT-1 is a distinct human Na(+)-coupled sulfate transporter, likely to play a major role in sulfate incorporation in HEV.

Amino Acid Sequence↗
PubMed2003

[Functional analysis of SIR2].

The yeast silent information regulator 2 (Sir2) is an NAD-dependent histone deacetylase and silences transcription at the mating type loci, telomeres and the ribosomal DNA. Over-expression of Sir2 extends the life span of yeast and C. elegans, while Sir2 knockout shortened yeast life span to about 50%. Mammalian cells also express several Sir2 homologues. However, most of physiological functions of Sir2 homologues seem to be unknown. We found that Sir2 alpha (SIRT1) was highly expressed during embryogenesis. In the adult mouse brain, ependymal cells and some cells of subventricular zone expressed Sir2 alpha. To identify proliferating neural cells in the adult brain, BrdU was administered in the drinking water for 2 weeks. BrdU-positive and nestin-positive cells expressed Sir2 alpha. Furthermore, neurosphere cultured from E14 mouse striatum expressed Sir2 alpha. The expression of Sir2 alpha in neurosphere disappeared after differentiation. Nicotinamide, splitomicin and sirtinol, potent inhibitors of Sir2 alpha, inhibited the growth of neurosphere. FACS analysis showed that sirtinol did not increase sub-G1 population of cells. Differentiation of neurosphere into neuron and oligodendrocyte was inhibited by the addition of nicotinamide, splitomicin or sirtinol in the differentiation medium. These results suggest that Sir2 alpha has a pivotal role in the proliferation and differentiation of neural stem cells.

Animals↗
PubMed2001

Functional analysis of the putative peroxidase domain of FANCA, the Fanconi anemia complementation group A protein.

Fanconi anemia (FA) is an autosomal recessive disorder manifested by chromosomal breakage, birth defects, and susceptibility to bone marrow failure and cancer. At least seven complementation groups have been identified, and the genes defective in four groups have been cloned. The most common subtype is complementation group A. Although the normal functions of the gene products defective in FA cells are not completely understood, a clue to the function of the FA group A gene product (FANCA) was provided by the detection of limited homology in the amino terminal region to a class of heme peroxidases. We evaluated this hypothesis by mutagenesis and functional complementation studies. We substituted alanine residues for the most conserved FANCA residues in the putative peroxidase domain and tested their effects on known biochemical and cellular functions of FANCA. While the substitution mutants were comparable to wild-type FANCA with regard to their stability, subcellular localization, and interaction with FANCG, only the Trp(183)-to-Ala substitution (W183A) abolished the ability of FANCA to complement the sensitivity of FA group A cells to mitomycin C. By contrast, TUNEL assays for apoptosis after exposure to H2O2 showed no differences between parental FA group A cells, cells complemented with wild-type FANCA, and cells complemented with the W183A of FANCA. Moreover, semiquantitative RT-PCR analysis for the expression of the peroxide-sensitive heme oxygenase gene showed appropriate induction after H2O2 exposure. Thus, W183A appears to be essential for the in vivo activity of FANCA in a manner independent of its interaction with FANCG. Moreover, neither wild-type FANCA nor the W183A mutation appears to alter the peroxide-induced apoptosisor peroxide-sensing ability of FA group A cells.

Alanine↗
PubMed2006

Structural and functional analysis of the pre-pore and membrane-inserted pore of Cry1Ab toxin.

Bacillus thuringiensis produces insecticidal Cry proteins that are active against different insect species. The primary action of Cry toxins is to lyse midgut epithelial cells in the target insect by forming lytic pores on the apical membrane. After interaction with cadherin receptor, Cry proteins undergo conformational changes from a monomeric structure to a pre-pore-oligomeric form that is able to interact with a second GPI-anchored aminopeptidase-N receptor and then insert into lipid membranes. Here, we review the recent advances in the understanding of the structural changes presented by Cry1Ab toxin upon membrane insertion. Based on analysis of the Trp fluorescence of pure monomeric and oligomeric Cry1Ab structures in solution and in membrane-bound state we reported that oligomerization caused 27% reduction of Trp exposed to the solvent. After membrane insertion there is another conformational change that allows an additional rearrangement of the Trp residues resulting in a total protection of these residues from exposure to the solvent. The oligomeric structure is membrane insertion competent since more than 96% of the Cry1Ab oligomer inserts into the membrane as a function of lipid:protein ratio, in contrast to the monomer of which only 5-10%, inserts into the membrane. Finally, analysis of the stability of monomeric, pre-pore and pore structures of Cry1Ab toxin after urea and thermal denaturation suggested that a more flexible conformation could be necessary for membrane insertion and this flexible structure is obtained by toxin oligomerization and by alkaline pH. Domain I is involved in the intermolecular interaction within the oligomeric Cry1Ab and this domain is inserted into the membrane in the membrane-inserted state.

Bacillus thuringiensis↗
PubMed2001

Molecular cloning and functional analysis of mouse C-terminal kinesin motor KifC3.

Proteins of the kinesin superfamily define a class of microtubule-dependent motors that play crucial roles in cell division and intracellular transport. To study the molecular mechanism of intracellular transport involving microtubule-dependent motors, a cDNA encoding a new kinesin-like protein called KifC3 was cloned from a mouse brain cDNA library. Sequence and secondary structure analysis revealed that KifC3 is a member of the C-terminal motor family. In contrast to other mouse C-terminal motors, KifC3 is apparently ubiquitous and may have a general role in intracellular transport. To understand the in vivo function of the KifC3 gene, we used homologous recombination in embryonic stem cells to construct knockout mouse strains for the KifC3 gene. Homozygous mutants of the KifC3 gene are viable, reproduce normally, and apparently develop normally. These results suggest that KifC3 is dispensable for normal development and reproduction in the mouse.

Amino Acid Sequence↗
PubMed1990

Cloning and functional analysis of a gene encoding a novel muscarinic acetylcholine receptor expressed in chick heart and brain.

Previous studies have demonstrated that muscarinic acetylcholine receptors (mAChR) expressed in chick heart are pharmacologically, immunologically, and biochemically distinct from mAChR expressed in mammalian heart. A chicken genomic clone encoding a mAChR whose deduced amino acid sequence is most homologous to the mammalian m4 receptor has been isolated. Northern blot analysis demonstrated that this gene is expressed in both chick heart and brain. The receptor encoded by this gene was expressed in stably transfected Chinese hamster ovary (CHO) and Y1 adrenal carcinoma cells in order to examine its ligand binding and functional properties. The receptor expressed in CHO and Y1 cells exhibits high affinity binding for the muscarinic antagonists quinuclidinyl benzilate and atropine, as well as the M1-selective antagonist pirenzepine and the M2-selective antagonist AF-DX 116. Therefore, when expressed in two heterologous cell lines, the cloned chick m4 receptor exhibits pharmacological properties similar to those previously reported for the chick cardiac receptor. This m4 receptor was able to mediate both agonist-dependent inhibition of forskolin-stimulated cAMP accumulation and agonist-dependent stimulation of phosphoinositide metabolism when expressed in CHO cells. In contrast, when expressed in Y1 cells, the chick m4 receptor mediated agonist-dependent inhibition of forskolin-stimulated cAMP accumulation, but not stimulation of phosphoinositide metabolism. Thus, as with the mammalian cardiac (m2) receptor, the functional specificity of the chick cardiac receptor appears to be dependent on the cell type in which it is expressed.

Amino Acid Sequence↗
PubMed2002

Development of a second generation monoclonal single chain variable fragment antibody against Venezuelan equine encephalitis virus: expression and functional analysis.

We have generated a single chain variable fragment (ScFv) antibody from a well-characterized monoclonal antibody (MAb) against Venezuelan equine encephalitis virus (VEE), by cloning variable regions of the heavy (V(H)) and the light (V(L)) chain antibody genes, connected by a DNA linker, in phagemid expression vector pCANTAB 5 E. MAb 1A4A1 was successfully cloned as a ScFv in Escherichia coli strain TG-1 and expressed as a approximately 30 kDa ScFv protein which was functional in recognizing VEE by ELISA. Results were reproduced in Escherichia coli strain HB2151 where the same clone, designated A116, was expressed primarily as soluble periplasmic protein. The 30 kDa A116 antibody displayed weak binding specificity to VEE antigen. Sequence analysis revealed a frame shift in the N-terminal region of the V(L) domain, upstream to the complementarity-determining region 1 (CDR1), as the probable cause of reduced activity. The protein sequence of A116 was highly homologous to published murine ScFv protein sequences except in the region of the identified frame shift.

Amino Acid Sequence↗
PubMed1996

Sequencing and functional analysis of the genome of Bacillus subtilis strain 168.

The international programme to sequence the 4.2 Mb genome of Bacillus subtilis, a model Gram-positive bacterium, is a joint project involving European, Japanese and US research groups. To date ca. 3.0 Mb of the genome has been sequenced, with the remaining 1.2 Mb expected to be completed in 1997. The amenability of B.subtilis to genetic manipulation, combined with the availability of extensive expertise on its biochemistry and physiology, makes this bacterium a valuable organism in which to investigate the properties of genes for which functions cannot be readily ascribed by standard methods.

Bacillus subtilis↗
PubMed1994

Functional analysis of the placenta-specific enhancer of the human glycoprotein hormone alpha subunit gene. Emergence of a new element.

Placental expression of the alpha subunit gene of the human glycoprotein hormones requires a multicomponent enhancer composed of tandem cAMP-response elements and an adjacent upstream regulatory element. Based on recent studies indicating that the upstream regulatory element includes binding sites for more than one protein, we investigated how functional activity correlated with these binding sites. Through extensive replacement mutagenesis of the native promoter regulatory region, we provide the first functional map of the upstream regulatory element. Within this region, we find that distinct proteins interact with three overlapping binding sites. While each site is functionally significant, no single site is essential or displays clear dominance. This is surprising since one of the sites binds a placenta-specific protein that heretofore has been regarded as essential for activity of the human alpha subunit placenta-specific enhancer. Consequently, our refined functional map of the upstream regulatory element reveals a complex combinatorial code that directs expression of the human alpha subunit gene to placenta.

Animals↗
PubMed1992

Functional analysis of peripheral blood lymphocytes isolated from patients with chronic hepatitis type B.

Cell-mediated immunity, evaluated by lymphocyte proliferation and expression of the activation antigen interleukin-2 receptor in response to mitogens such as phytohemagglutinin and concanavalin-A, has been reported to be defective in chronic hepatitis B virus carriers. However, no definite conclusion on the functional state of T cells from these patients can be drawn. In the present study, we have investigated the expression of a wide set of lymphoid activation molecules as well as the proliferative response of peripheral blood lymphocytes isolated from patients with chronic hepatitis type B after in vitro stimulation with monoclonal antibodies to both the T-cell receptor-CD3 complex and the CD2 molecule, which are the two main T-cell activation pathways. Our findings show that peripheral T lymphocytes from patients with chronic hepatitis type B express the activation antigens 4F2 molecule, interleukin-2 receptor, and activation inducer molecule (AIM) antigen, and proliferate normally after specific stimulation through either the T-cell receptor-CD3 complex or the CD2 molecule. These results suggest that the peripheral blood T cells of patients with chronic hepatitis B are fully operative and functionally competent in vitro.

Adult↗
PubMed1987

Structural and functional analysis of a human 7 S K RNA gene.

Using purified RNA from HeLa cells, we have synthesized and cloned a cDNA encoding an almost entire 7 S K RNA. This cDNA probe was used to isolate 7 S K RNA gene sequences from a human genomic library by high-stringency colony hybridization. In order to differentiate between functional genes and related sequences, we have used a rapid in-vitro transcription assay of purified phage DNA. With this additional screening criterion applied to selected clones, we have obtained one recombinant phage that contained a complete 7 S K RNA gene and, immediately adjacent to its 3' end, a truncated pseudogene. The nucleotide sequence of both genes including the flanking regions has been determined. The functional integrity of the isolated 7 S K RNA gene was verified by in-vitro transcription studies with cell-free extracts and by fingerprinting of the specific transcripts with ribonuclease T1. Under optimal ionic conditions, the transcription efficiency in vitro of this 7 S K RNA gene was found to be comparable to that of a human 7 S K RNA in vitro depends on 5'-flanking sequences. The region up to position -67 was determined to be essential for efficient transcription in vitro of 7 S K RNA. While apparently a variety of 7 S K related sequences is distributed within the human genome, hybridization of 5'-flanking sequences to genomic DNA revealed that possibly not more than one copy of this gene is present per haploid genome.

Base Sequence↗
PubMed1998

Systematic functional analysis of the yeast genome.

The genome sequence of the yeast Saccharomyces cerevisiae has provided the first complete inventory of the working parts of a eukaryotic cell. The challenge is now to discover what each of the gene products does and how they interact in a living yeast cell. Systematic and comprehensive approaches to the elucidation of yeast gene function are discussed and the prospects for the functional genomics of eukaryotic organisms evaluated.

Genetic Techniques↗

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