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M Kessel

Publications and source records attributed to M Kessel.

At least 37 records · Page 2Linked to original sources
PubMed1998

Molecular properties of ClpAP protease of Escherichia coli: ATP-dependent association of ClpA and clpP.

The ClpAP protease from Escherichia coli consists of the ATP-binding regulatory component, ClpA (subunit Mr 84 165), and the proteolytic component, ClpP (subunit Mr 21 563). Our hydrodynamic studies demonstrate that the predominant forms of these proteins in solution correspond to those observed by electron microscopy. ClpP and proClpP(SA), which in electron micrographs appear to have subunits arranged in rings of seven subunits, were found by ultracentrifugation to have s20,w values of 12.2 and 13.2 S and molecular weights of 300 000 and 324 000 +/- 3000, respectively, indicating that the native form of each consists of two such rings. The two intact rings of ClpP were separated in the presence of >/= 0.1 M sulfate at low temperatures, suggesting that ring-ring contacts are polar in nature and more easily disrupted than subunit contacts within individual rings. Sedimentation equilibrium analysis indicated that ClpA purified without nucleotide exists as an equilibrium mixture of monomers and dimers with Ka = (1.0 +/- 0.2) x 10(5) M-1 and that, upon addition of MgATP or adenosine 5'-O-(3-thiotriphosphate), ClpA subunits associated to a form with Mr 505 000 +/- 5000, consistent with the hexameric structure seen by electron microscopy. Sedimentation velocity and gel-filtration analysis showed that the nucleotide-promoted hexamer of ClpA (s20,w = 17.2 S) binds tightly to ClpP producing species with s20,w values of 21 and 27 S (f/f0 = 1.5 and 1.8, respectively), consistent with electron micrographs of ClpAP that show a single tetradecamer of ClpP associated with either one or two ClpA hexamers [Kessel et al. (1995) J. Mol. Biol. 250, 587-594]. Under assay conditions in the presence of ATP and Mg2+, the apparent dissociation constant of hexameric ClpA and tetradecameric ClpP was approximately 4 +/- 2 nM. By the method of continuous variation, the optimal ratio of ClpA to ClpP in the active complex was 2:1. The specific activities of limiting ClpA and ClpP determined in the presence of an excess of the other component indicated that the second molecule of ClpA provides very little additional activation of ClpP.

Adenosine Triphosphatases↗
PubMed1998

Enzymatic and structural similarities between the Escherichia coli ATP-dependent proteases, ClpXP and ClpAP.

Escherichia coli ClpX, a member of the Clp family of ATPases, has ATP-dependent chaperone activity and is required for specific ATP-dependent proteolytic activities expressed by ClpP. Gel filtration and electron microscopy showed that ClpX subunits (Mr 46, 000) associate to form a six-membered ring (Mr approximately 280, 000) that is stabilized by binding of ATP or nonhydrolyzable analogs of ATP. ClpP, which is composed of two seven-membered rings stacked face-to-face, interacts with the nucleotide-stabilized hexamer of ClpX to form a complex that could be isolated by gel filtration. Electron micrographs of negatively stained ClpXP preparations showed side views of 1:1 and 2:1 ClpXP complexes in which ClpP was flanked on either one or both sides by a ring of ClpX. Thus, as was seen for ClpAP, a symmetry mismatch exists in the bonding interactions between the seven-membered rings of ClpP and the six-membered rings of ClpX. Competition studies showed that ClpA may have a slightly higher affinity (approximately 2-fold) for binding to ClpP. Mixed complexes of ClpA, ClpX, and ClpP with the two ATPases bound simultaneously to opposite faces of a single ClpP molecule were seen by electron microscopy. In the presence of ATP or nonhydrolyzable analogs of ATP, ClpXP had nearly the same activity as ClpAP against oligopeptide substrates (>10,000 min-1/tetradecamer of ClpP). Thus, ClpX and ClpA interactions with ClpP result in structurally analogous complexes and induce similar conformational changes that affect the accessibility and the catalytic efficiency of ClpP active sites.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1998

At sixes and sevens: characterization of the symmetry mismatch of the ClpAP chaperone-assisted protease.

ClpAP, a typical energy-dependent protease, consists of a proteolytic component (ClpP) and a chaperone-like ATPase (ClpA). ClpP is composed of two apposed heptameric rings, whereas in the presence of ATP or ATPgammaS, ClpA is a single hexameric ring. Formation of ClpAP complexes involves a symmetry mismatch as sixfold ClpA stacks axially on one or both faces of sevenfold ClpP. We have analyzed these structures by cryo-electron microscopy. Our three-dimensional reconstruction of ClpA at 29-A resolution shows the monomer to be composed of two domains of similar size that, in the hexamer, form two tiers enclosing a large cavity. Cylindrical reconstruction of ClpAP reveals three compartments: the digestion chamber inside ClpP; a compartment between ClpP and ClpA; and the cavity inside ClpA. They are connected axially via narrow apertures, implying that substrate proteins should be unfolded to allow translocation into the digestion chamber. The cavity inside ClpA is structurally comparable to the "Anfinsen cage" of other chaperones and may play a role in the unfolding of substrates. A geometrical description of the symmetry mismatch was obtained by using our model of ClpA and the crystal structure of ClpP (Wang et al., 1997, Cell 91, 447-456) to identify the particular side views presented by both molecules in individual complexes. The interaction is characterized by a key pair of subunits, one of each protein. A small turn (8.6(o) = 2pi/42; equivalent to a 4-A shift) would transfer the key interaction to another pair of subunits. We propose that nucleotide hydrolysis results in rotation, facilitating the processive digestion of substrate proteins.

Adenosine Triphosphatases↗
PubMed1998

Demarcation of ventral territories by the homeobox gene NKX2.1 during early chick development.

Members of the NK-2 homeobox gene family are expressed in distinct parts of the central nervous system and in other non-neural territories not only in the fruitfly Drosophila melanogaster, but also in vertebrates. The murine Nkx2.1 (TTF-1, T/ebp) gene was previously shown to be active and indispensable in the developing forebrain, hypophysis, thyroid and lung. Here we report the early transcript distribution of the chick NKX2.1 gene. By whole-mount in situ hybridization we detect a novel transient expression domain in the early epiblast. Further expression occurs in the ventral medial endoderm, which becomes restricted to the anlage fields of the thyroid and lung, in the ventral diencephalon and telencephalon. These findings suggest that NKX2.1 is part of a Nkx code which specifies ventral territories in the vertebrate embryo.

Animals↗
PubMed1998

CMIX, a paired-type homeobox gene expressed before and during formation of the avian primitive streak.

We cloned a chicken homeobox gene closely related to the Xenopus Mix. 1 gene. CMIX is expressed early in embryogenis in a sickle-shaped area in the posterior zone of the blastoderm. With the beginning of gastrulation, CMIX transcripts are found in the primitive streak primordium, then in the young and medium-sized streak, however not in the mesoderm after its emergence. In the fully-extended streak, CMIX is restricted to its middle, i.e. the prospective ventral mesoderm. CMIX RNA is undetectable by whole-mount in-situ analysis in later stages. We compare CMIX expression to the early pattern of the brachyury gene.

Amino Acid Sequence↗
PubMed1998

Halogeometricum borinquense gen. nov., sp. nov., a novel halophilic archaeon from Puerto Rico.

A novel extremely halophilic archaeon was isolated from the solar salterns of Cabo Rojo, Puerto Rico. The organism is very pleomorphic, motile and requires at least 8% (w/v) NaCl to grow. Polar lipid composition revealed the presence of a novel non-sulfate-containing glycolipid and the absence of the glycerol diether analogue of phosphatidylglycerosulfate. The G + C content of the DNA is 59 mol%. On the basis of 16S rRNA sequence data, the new isolate cannot be classified in one of the recognized genera, but occupies a position that is distantly related to the genus Haloferax. All these features justify the creation of a new genus and a new species for the family Halobacteriaceae, order Halobacteriales. The name Halogeometricum borinquense gen. nov., sp. nov. is proposed. The type strain is ATCC 700274T.

Base Composition↗
PubMed1997

Multiple conformational states of the bacteriophage T4 capsid surface lattice induced when expansion occurs without prior cleavage.

The maturation pathway of bacteriophage T4 capsid provides a model system for the study of largescale conformational changes, in that the precursor capsid progresses through four long-lived and widely differing states. The surface lattice first assembled (uncleaved/unexpanded state: hexagonal lattice constant, a = 11.8 nm) undergoes proteolytic cleavage (cleaved/unexpanded state), then expands (cleaved/ expanded state: a = 14.0 nm), and then binds accessory proteins. The most profound change, expansion, normally follows cleavage of the major capsid protein gp23 to gp23* (the 65-residue N-terminal "delta-domain" is removed), but can be induced in vitro in the absence of cleavage by treatment with 0.25 M guanidine-HCl (uncleaved/expanded state). We have studied this alternative pathway by negative staining electron microscopy of polyheads (tubular capsid variants). We find that uncleaved/expanded polyheads encompass four discrete states, called G1-G4, distinguished by their lattice constants of 12.6 nm (G1), 13.4 nm (G2), and 14.0 nm (G3, G4) and by the structures of their hexameric capsomers. Viewed in projection, the G4 capsomer differs from the cleaved/ expanded capsomer only in the presence of additional mass at one site per protomer. This mass correlates with the presence of the delta-domain, which translocates from the inner to the outer surface when the uncleaved lattice expands. Based on proximity of resemblance among these capsomers, we suggest that G1 to G4 represent a sequence of transitional states whose endpoint is G4. G1, G2, and G3 may correspond to intermediates that are too short-lived to be observed when the cleaved lattice expands, but are trapped by the retention of delta-domains at the interfaces between subunits in the uncleaved lattice.

Bacteriophage T4↗
PubMed1997

Gastrulation and homeobox genes in chick embryos.

We review the early stages of chick embryogenesis, in particular the formation of the hypoblast, and the ingression of endoderm and mesoderm through the primitive streak. The formation of a trilaminar embryo during gastrulation is accompanied by the specification of body axes. The first axis is already present in the unfertilized egg and runs from the cytoplasmatic animal to the yolk rich vegetal pole. Already within the uterus a second axis conveys bilateral symmetry to the embryo. It extends from a dorsal/anterior to a ventral/posterior position. These axial poles segregate during gastrulation to form the classical coordinates, a dorsal-ventral and an anterior-posterior axis. The establishment of axes is accompanied by the expression of specific combinations of homeobox genes during gastrulation in the chick, as in other metazoa. We review the avian specific information and compare it with findings in other species. A combinatorial homeobox code for the specification of identities during development is discussed.

Animals↗
PubMed1997

Proteolysis of the phage lambda CII regulatory protein by FtsH (HflB) of Escherichia coli.

Rapid proteolysis plays an important role in regulation of gene expression. Proteolysis of the phage lambda CII transcriptional activator plays a key role in the lysis-lysogeny decision by phage lambda. Here we demonstrate that the E. coli ATP-dependent protease FtsH, the product of the host ftsH/hflB gene, is responsible for the rapid proteolysis of the CII protein. FtsH was found previously to degrade the heat-shock transcription factor sigma32. Proteolysis of sigma32 requires, in vivo, the presence of the DnaK-DnaJ-GrpE chaperone machine. Neither DnaK-DnaJ-GrpE nor GroEL-GroES chaperone machines are required for proteolysis of CII in vivo. Purified FtsH carries out specific ATP-dependent proteolysis of CII in vitro. The degradation of CII is at least 10-fold faster than that of sigma32. Electron microscopy revealed that purified FtsH forms ring-shaped structures with a diameter of 6-7 nm.

ATP-Dependent Proteases↗
PubMed1997

Fine-structure evidence for cell membrane partitioning of the nucleoid and cytoplasm during bud formation in Hyphomonas species.

Hyphomonas spp. reproduce by budding from the tip of the prosthecum, distal to the main body of the reproductive cell; thus, the chromosome must travel through the prosthecum to enter the progeny, the swarm cell. When viewed by electron microscopy, negatively stained whole cells, ultrathin-sectioned cells, and freeze-etched and frozen hydrated cells all had marked swellings of the cytoplasmic membrane (CM) in the prosthecum which are termed pseudovesicles (PV). PV were separated by constrictions in the contiguous CM. In replicating cells, PV housed ribosomes and DNA, which was identified by its fibrillar appearance and by lactoferrin-gold labeling. The micrographs also revealed that the CM bifurcates at the origin of the prosthecum so that one branch partitions the main body of the reproductive cell from the prosthecum and swarm cell. The results of this fine-structure analysis suggest models explaining DNA segregation and the marked asymmetric polarity of the budding reproductive cell.

Cell Division↗
PubMed1997

Patterning of the chick forebrain anlage by the prechordal plate.

We analysed the role of the prechordal plate in forebrain development of chick embryos in vivo. After transplantation to uncommitted ectoderm a prechordal plate induces an ectopic, dorsoventrally patterned, forebrain-like vesicle. Grafting laterally under the anterior neural plate causes ventralization of the lateral side of the forebrain, as indicated by a second expression domain of the homeobox gene NKX2.1. Such a lateral ventralization cannot be induced by the secreted factor Sonic Hedgehog alone, as this is only able to distort the ventral forebrain medially. Removal of the prechordal plate does not reduce the rostrocaudal extent of the anterior neural tube, but leads to significant narrowing and cyclopia. Excision of the head process results in the caudal expansion of the NKX2.1 expression in the ventral part of the anterior neural tube, while PAX6 expression in the dorsal part remains unchanged. We suggest that there are three essential steps in early forebrain patterning, which culminate in the ventralization of the forebrain. First, anterior neuralization occurs at the primitive streak stage, when BMP-4-antagonizing factors emanate from the node and spread in a planar fashion to induce anterior neural ectoderm. Second, the anterior translocation of organizer-derived cells shifts the source of neuralizing factors anteriorly, where the relative concentration of BMP-4-antagonists is thus elevated, and the medial part of the prospective forebrain becomes competent to respond to ventralizing factors. Third, the forebrain anlage is ventralized by signals including Sonic Hedgehog, thereby creating a new identity, the prospective hypothalamus, which splits the eye anlage into two lateral domains.

Animals↗
PubMed1997

Segregating expression domains of two goosecoid genes during the transition from gastrulation to neurulation in chick embryos.

We report the isolation and characterization of a chicken gene, GSX, containing a homeobox similar to that of the goosecoid gene. The structure of the GSX gene and the deduced GSX protein are highly related to the previously described goosecoid gene. The two homeodomains are 74% identical. In the first few hours of chick embryogenesis, the expression pattern of GSX is similar to GSC, in the posterior margin of the embryo and the young primitive streak. Later during gastrulation, expression of the two genes segregate. GSC is expressed in the anterior part of the primitive streak, then in the node, and finally in the pre-chordal plate. GSX is expressed in the primitive streak excluding the node, and then demarcating the early neural plate around the anterior streak and overlying the pre-chordal plate. We demonstrate that the GSX-positive part of the primitive streak induces gastrulation, while the GSC-expressing part induces neurulation. After full extension of the streak, the fate of cells now characterized by GSX is to undergo neurulation, while those expressing GSC undergo gastrulation. We discuss the effect of a duplicated basic goosecoid identity for the generation of a chordate nervous system in ontogeny and phylogeny.

Amino Acid Sequence↗
PubMed1996

Differential activation of the clustered homeobox genes CNOT2 and CNOT1 during notogenesis in the chick.

CNOT2, a newly identified homeobox gene, is physically linked to the CNOT1 gene in the chicken genome. The two chicken genes represent two different subgroups of the Not gene family, the first including CNOT1 and the Xenopus genes XNot1 and XNot2, and the second CNOT2 and the zebrafish floating head gene. The overall expression pattern of CNOT2 in Hensen's node, notochord, neural plate, tailbud, and epiphysis resembled the CNOT1 pattern. However, several significant differences occurred: CNOT2 expression was much stronger and more widespread in the pregastrulation embryo, it showed an additional, transient domain on the anterior intestinal portal, and lacked expression on the early anterior neural folds and the anterodistal limb bud. We studied CNOT expression by transplanting parts of the primitive streak into growing embryos or by explanting them into tissue culture. CNOT gene expression from young nodes was maintained in vivo, but required in vitro the addition of retinoic acid. The generation of differentiated notochord structures could only be obtained, if either older node grafts were used in vitro or young node grafts were transplanted close to the primary axis in vivo. We conclude that CNOT expression in the anterior streak is not enough for notochord differentiation, but further influences are necessary. A Not-related gene has previously been isolated from Drosophila melanogaster and its expression was detected in the posterior brain and the neuroblasts (Dessain and McGinnis, 1993. Adv. Dev. Biochem. 2, 1-55). The correspondence between Not gene-expressing cells in the nervous system of Drosophila and the early neuroectoderm in the chick and its implication for a phylogenetic relationship between neuroectoderm and the notochord is discussed.

Amino Acid Sequence↗
PubMed1996

Six-fold rotational symmetry of ClpQ, the E. coli homolog of the 20S proteasome, and its ATP-dependent activator, ClpY.

ClpQ (HslV) is a homolog of the beta-subunits of the 20S proteasome. In E. coli, it is expressed from an operon that also encodes ClpY (HslU), an ATPase homologous to the protease chaperone, ClpX. ClpQ (subunit Mr 19,000) and ClpY (subunit Mr 49,000) were purified separately as oligomeric proteins with molecular weights of approximately 220,000 and approximately 350,000, respectively, estimated by gel filtration. Mixtures of ClpY and ClpQ displayed ATP-dependent proteolytic activity against casein, and a complex of the two proteins was isolated by gel filtration in the presence of ATP. Image processing of negatively stained electron micrographs revealed strong six-fold rotational symmetry for both ClpY and ClpQ, suggesting that the subunits of both proteins are arranged in hexagonal rings. The molecular weight of ClpQ combined with its symmetry is consistent with a double hexameric ring, whereas the data on ClpY suggest only one such ring. The symmetry mismatch previously observed between hexameric ClpA and heptameric ClpP in the related ClpAP protease is apparently not reproduced in the symmetry-matched ClpYQ system.

ATP-Dependent Proteases↗
PubMed1996

Checklist: vertebrate homeobox genes.

Up to now around 170 different homeobox genes have been cloned from vertebrate genomes. A compilation of the various isolates from mouse, chick, frog, fish and man is presented in the form of a concise checklist, including the designations from the original publications. Putative homologs from different species are aligned, and key characteristics of embryonic or adult expression domains, as well as mutant phenotypes are briefly indicated.

Animals↗
PubMed1995

The protein-folding activity of chaperonins correlates with the symmetric GroEL14(GroES7)2 heterooligomer.

Chaperonins GroEL and GroES form, in the presence of ATP, two types of heterooligomers in solution: an asymmetric GroEL14GroES7 "bullet"-shaped particle and a symmetric GroEL14(GroES7)2 "football"-shaped particle. Under limiting concentrations of ATP or GroES, excess ADP, or in the presence of 5'-adenylyl imidodiphosphate, a correlation is seen between protein folding and the amount of symmetric GroEL14(GroES7)2 particles in a chaperonin solution, as detected by electron microscopy or by chemical crosslinking. Kinetic analysis suggests that protein folding is more efficient when carried out by a chaperonin solution populated with a majority of symmetric GroEL14(GroES7)2 particles than by a majority of asymmetric GroEL14GroES7 particles. The symmetric heterooligomer behaves as a highly efficient intermediate of the chaperonin protein folding cycle in vitro.

Adenosine Diphosphate↗