scholarly journals Identification of the chromosome localization domain of the Drosophila nod kinesin-like protein.

1995 ◽  
Vol 131 (4) ◽  
pp. 833-843 ◽  
Author(s):  
K Afshar ◽  
J Scholey ◽  
R S Hawley
Keyword(s):  
Meiotic Spindle ◽  
Mitotic Chromosomes ◽  
Chromosome Localization ◽  
Amino Terminal ◽  
Amino Acid Region ◽  
Meiotic Chromosomes ◽  
Subcellular Component ◽  
Carboxyl Terminal ◽  
Acid Region

The nod kinesin-like protein is localized along the arms of meiotic chromosomes and is required to maintain the position of achiasmate chromosomes on the developing meiotic spindle. Here we show that the localization of ectopically expressed nod protein on mitotic chromosomes precisely parallels that observed for wild-type nod protein on meiotic chromosomes. Moreover, the carboxyl-terminal half of the nod protein also binds to chromosomes when overexpressed in mitotic cells, whereas the overexpressed amino-terminal motor domain binds only to microtubules. Chromosome localization of the carboxyl-terminal domain of nod depends upon an 82-amino acid region comprised of three copies of a sequence homologous to the DNA-binding domain of HMG 14/17 proteins. These data map the two primary functional domains of the nod protein in vivo and provide a molecular explanation for the directing of the nod protein to a specific subcellular component, the chromosome.

Effects of truncated neurofilament proteins on the endogenous intermediate filaments in transfected fibroblasts

1991 ◽  
Vol 99 (2) ◽  
pp. 335-350 ◽  
Author(s):  
S.S. Chin ◽  
P. Macioce ◽  
R.K. Liem
Keyword(s):  
Intermediate Filament ◽  
Dominant Negative ◽  
Deletion Mutants ◽  
Tail Domain ◽  
Cultured Fibroblasts ◽  
Mutant Proteins ◽  
Amino Terminal ◽  
Novel Approach ◽  
Carboxyl Terminal

The expression and assembly characteristics of carboxyl- and amino-terminal deletion mutants of rat neurofilament low Mr (NF-L) and neurofilament middle Mr (NF-M) proteins were examined by transient transfection of cultured fibroblasts. Deletion of the carboxyl-terminal tail domain of either protein indicated that this region was not absolutely essential for co-assembly into the endogenous vimentin cytoskeleton. However, deletion into the alpha-helical rod domain resulted in an inability of the mutant proteins to co-assemble with vimentin into filamentous structures. Instead, the mutant proteins appeared to be assembled into unusual tubular-vesicular structures. Additionally, these latter deletions appeared to act as dominant negative mutants which induced the collapse of the endogenous vimentin cytoskeleton as well as the constitutively expressed NF-H and NF-M cytoskeletons in stably transfected cell lines. Thus, an intact alpha-helical rod domain was essential for normal IF co-assembly whereas carboxyl-terminal deletions into this region resulted in dramatic alterations of the existing type III and IV intermediate filament cytoskeletons in vivo. Deletions from the amino-terminal end into the alpha-helical rod region gave different results. With these deletions, the transfected protein was not co-assembled into filaments and the endogenous vimentin IF network was not disrupted, indicating that these deletion mutants are recessive. The dominant negative mutants may provide a novel approach to studying intermediate filament function within living cells.

scholarly journals The yeast silent information regulator Sir4p anchors and partitions plasmids.

1997 ◽  
Vol 17 (12) ◽  
pp. 7061-7068 ◽  
Author(s):  
A Ansari ◽  
M R Gartenberg
Keyword(s):  
Coiled Coil ◽  
Axial Rotation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Interacting Protein ◽  
Segregation Behavior ◽  
Amino Acid Region ◽  
Dividing Cells ◽  
Nuclear Component ◽  
Acid Region

Circular plasmids containing telomeric TG1-3 arrays or the HMR E silencer segregate efficiently between dividing cells of the yeast Saccharomyces cerevisiae. Subtelomeric X repeats augment the TG1-3 partitioning activity by a process that requires the SIR2, SIR3, and SIR4 genes, which are also required for silencer-based partitioning. Here we show that targeting Sir4p to DNA directly via fusion to the bacterial repressor LexA confers efficient mitotic segregation to otherwise unstable plasmids. The Sir4p partitioning activity resides within a 300-amino-acid region (residues 950 to 1262) which precedes the coiled-coil dimerization motif at the extreme carboxy end of the protein. Using a topology-based assay, we demonstrate that the partitioning domain also retards the axial rotation of LexA operators in vivo. The anchoring and partitioning properties of LexA-Sir4p chimeras persist despite the loss of the endogenous SIR genes, indicating that these functions are intrinsic to Sir4p and not to a complex of Sir factors. In contrast, inactivation of the Sir4p-interacting protein Rap1p reduces partitioning by a LexA-Sir4p fusion. The data are consistent with a model in which the partitioning and anchoring domain of Sir4p (PAD4 domain) attaches to a nuclear component that divides symmetrically between cells at mitosis; DNA linked to Sir4p by LexA serves as a reporter of protein movement in these experiments. We infer that the segregation behavior of telomere- and silencer-based plasmids is, in part, a consequence of these Sir4p-mediated interactions. The assays presented herein illustrate two novel approaches to monitor the intracellular dynamics of nuclear proteins.

In vivo effect of sodium orthovanadate on pp60c-src kinase

1987 ◽  
Vol 7 (3) ◽  
pp. 1139-1147
Author(s):  
J W Ryder ◽  
J A Gordon
Keyword(s):  
Tyrosine Kinase ◽  
Chicken Embryo ◽  
Sodium Orthovanadate ◽  
Normal Cells ◽  
Amino Terminal ◽  
Carboxyl Terminal ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

We have compared the tyrosine kinase activity of pp60c-src isolated from intact chicken embryo fibroblasts treated with micromolar sodium orthovanadate for 4 h and from untreated cells. We found an approximate 50% reduction in both autophosphorylation of pp60c-src and phosphorylation of casein when examined in the immune complex kinase assay. The reduction of in vitro enzymatic activity correlated with a vanadate-induced increase in in vivo phosphorylation of pp60c-src at the major site of tyrosine phosphorylation in the carboxyl-terminal half of the molecule and at serine in the amino-terminal half of the molecule. Our observations in vivo and those of Courtneidge in vitro (EMBO J. 4:1471-1477, 1985) suggest that vanadate may enhance a cellular regulatory mechanism that inhibits the activity of pp60c-src in normal cells. A likely candidate for this mechanism is phosphorylation at a tyrosine residue distinct from tyrosine 416, probably tyrosine 527 in the carboxyl-terminal sequence of amino acids unique to pp60c-src. The regulatory role, if any, of serine phosphorylation in pp60c-src remains unclear. The 36-kilodalton phosphoprotein, a substrate of pp60v-src, showed a significant phosphorylation at tyrosine after treatment of normal chicken embryo fibroblasts with vanadate. Assuming that pp60c-src is inhibited intracellularly by vanadate, either another tyrosine kinase is stimulated by vanadate (e.g., a growth factor receptor) or the 36-kilodalton phosphoprotein in normal cells is no longer rapidly dephosphorylated by a tyrosine phosphatase in the presence of vanadate.

Purification of human procollagen type I carboxyl-terminal propeptide cleaved as in vivo from procollagen and used to calibrate a radioimmunoassay of the propeptide

1994 ◽  
Vol 40 (5) ◽  
pp. 811-816 ◽  
Author(s):  
B J Pedersen ◽  
M Bonde
Keyword(s):  
Geometric Mean ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Type I ◽  
Procollagen Type ◽  
Amino Terminal ◽  
Fetal Fibroblasts ◽  
Procollagen Type I ◽  
Carboxyl Terminal

Abstract We purified human procollagen type I carboxyl-terminal propeptide (PICP) that had been cleaved as in vivo from procollagen. PICP in serum-free medium from cultured human fetal fibroblasts was purified by thiophilic adsorption chromatography, low-pressure gel filtration, and HPLC gel filtration. The purity and homogeneity of the protein was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal amino acid sequencing showed that the sequences of the alpha 1 and alpha 2 chains of this PICP were identical to those of the PICP produced in vivo. The monocomponent PICP thus purified was used as calibrator in a simple equilibrium-type RIA of PICP with polyclonal antibodies raised in rabbits. The measuring range is 0.15-3.75 nmol/L, and the assay detection limit is 0.03 nmol/L. The within-run and total CVs are 2% and 4%, respectively. The reference interval for the plasma concentration of PICP in healthy women of ages > 30 years is 0.36-1.44 nmol/L (geometric mean 0.72 nmol/L, n = 154).

scholarly journals A Carboxy-Terminal 16-Amino-Acid Region of ς38 ofEscherichia coli Is Important for Transcription under High-Salt Conditions and Sigma Activities In Vivo

2000 ◽  
Vol 182 (16) ◽  
pp. 4628-4631 ◽  
Author(s):  
Mio Ohnuma ◽  
Nobuyuki Fujita ◽  
Akira Ishihama ◽  
Kan Tanaka ◽  
Hideo Takahashi
Keyword(s):  
Amino Acid ◽  
Bacterial Species ◽  
High Potassium ◽  
Transcription Analysis ◽  
Amino Acid Region ◽  
Sigma Subunit ◽  
Carboxy Terminal ◽  
Acid Region

ABSTRACT ς38 (or ςS, the rpoS gene product) is a sigma subunit of RNA polymerase in Escherichia coli and directs transcription from a number of stationary-phase promoters as well as osmotically inducible promoters. In this study, we analyzed the function of the carboxy-terminal 16-amino-acid region of ς38 (residues 315 to 330), which is well conserved among the rpoS gene products of enteric bacterial species. Truncation of this region was shown to result in the loss of sigma activity in vivo using promoter-lacZ fusion constructs, but the mutant ς38 retained the binding activity in vivo to the core enzyme. The in vitro transcription analysis revealed that the transcription activity of ς38 holoenzyme under high potassium glutamate concentrations was significantly decreased by the truncation of the carboxy-terminal tail element.

scholarly journals Overexpression of C-terminally but not N-terminally truncated Myb induces fibrosarcomas: a novel nonhematopoietic target cell for the myb oncogene.

1994 ◽  
Vol 14 (4) ◽  
pp. 2278-2290 ◽  
Author(s):  
R D Press ◽  
E P Reddy ◽  
D L Ewert
Keyword(s):  
Amino Acid Sequences ◽  
Hematopoietic Cell ◽  
Full Spectrum ◽  
Truncated Protein ◽  
Amino Terminal ◽  
Myb Gene ◽  
Integration Sites ◽  
Carboxyl Terminal ◽  
Cell Transforming

The myb oncogene encodes a DNA-binding transcriptional transactivator which can become a hematopoietic cell-transforming protein following the deletion of amino acid sequences from either its amino or carboxyl terminus. Although a number of hematopoietic tumors express terminally deleted variants of Myb, the involvement of truncated Myb in nonhematopoietic tumors has not been adequately investigated. To assess the full spectrum of Myb's oncogenic capability, a replication-competent retroviral vector (RCAMV) was used to express a full-length protein (C-Myb), an amino-terminally truncated protein (VCC- or delta N-Myb), a carboxyl-terminally truncated protein (T-Myb), or a doubly truncated protein (VCT-Myb) in vivo. These viruses were injected intravenously into 10-day chicken embryos, and the infected chicks were monitored for tumors. Approximately 4 to 8 weeks after hatching, the majority (30 of 39 [77%]) of animals infected with the T-Myb retrovirus (without 214 carboxyl-terminal residues) developed nodular muscle tumors which could be identified by both morphologic and immunohistochemical criteria as fibrosarcomas. Identically appearing tumors could also be found in the kidney of some T-Myb-infected animals. The T-Myb-induced fibrosarcomas expressed the appropriately sized T-Myb protein, contained an unaltered proviral T-myb gene, and showed clonal proviral integration sites. In comparison, no sarcomas were observed in any of the animals infected with the amino-terminally truncated (VCC- and delta N-Myb) or doubly truncated (VCT-Myb) viruses. A loss of carboxyl-terminal but not amino-terminal sequences can thus convert Myb into a potent in vivo transforming protein for nonhematopoietic mesenchymal cells. In comparison, a truncation of either or both ends of the protein can activate Myb into a hematopoietic cell-transforming protein.

scholarly journals Sequence and functional analysis of the positively acting regulatory gene amdR from Aspergillus nidulans.

1990 ◽  
Vol 10 (6) ◽  
pp. 3194-3203 ◽  
Author(s):  
A Andrianopoulos ◽  
M J Hynes
Keyword(s):  
Dna Binding ◽  
Aspergillus Nidulans ◽  
Nuclear Localization ◽  
Regulatory Gene ◽  
Transcription Activation ◽  
Binding Motif ◽  
Wild Type ◽  
Amino Terminal ◽  
Carboxyl Terminal

The positively acting regulatory gene amdR of Aspergillus nidulans coordinately regulates the expression of five structural genes involved in the catabolism of certain amides (amdS), omega amino acids (gatA and gabA), and lactams (lamA and lamB) in the presence of omega amino acid inducers. Analysis of the amdR gene showed that it contains three small introns, heterogeneous 5' and 3' transcription sites, and multiple AUG codons prior to the major AUG initiator. The predicted amdR protein sequence has a cysteine-rich "zinc finger" DNA-binding motif at the amino-terminal end, four putative acidic transcription activation motifs in the carboxyl-terminal half, and two sequences homologous to the simian virus 40 large T antigen nuclear localization motif. These nuclear localization sequences overlap the cysteine-rich DNA-binding motif. A series of 5', 3', and internal deletions were examined in vivo for transcription activator function and showed that the amdR product contains at least two activation regions in the carboxyl-terminal half. Each of these activator amdR product contains at least two activation regions in the carboxyl-terminal half. Each of these activator regions may function independently, but both are required for wild-type levels of transcription activation. A number of the amdR deletion products were found to compete with the wild-type amdR product in vivo. Development of a rapid method for the localization of amdR mutations is presented, and using this technique, we localized and sequenced the mutation in the semiconstitutive amdR6c allele. The amdR6c missense mutation occurs in the middle of the gene, and it is suggested that it results in an altered protein which activates gene expression efficiently in the absence of an inducer.

scholarly journals Reexamination of the Role of the Amino Terminus of SecA in Promoting Its Dimerization and Functional State

2008 ◽  
Vol 190 (21) ◽  
pp. 7302-7307 ◽  
Author(s):  
Sanchaita Das ◽  
Elizabeth Stivison ◽  
Ewa Folta-Stogniew ◽  
Donald Oliver
Keyword(s):  
Cell Growth ◽  
Functional State ◽  
Protein Translocation ◽  
Specific Activity ◽  
Cell Fractionation ◽  
Mutant Proteins ◽  
Amino Terminal ◽  
Carboxyl Terminal

ABSTRACT The SecA nanomotor promotes protein translocation in eubacteria by binding both protein cargo and the protein-conducting channel and by undergoing ATP-driven conformation cycles that drive this process. There are conflicting reports about whether SecA functions as a monomer or dimer during this dynamic process. Here we reexamined the roles of the amino and carboxyl termini of SecA in promoting its dimerization and functional state by examining three secA mutants and the corresponding proteins: SecAΔ8 lacking residues 2 to 8, SecAΔ11 lacking residues 2 to 11, and SecAΔ11/N95 lacking both residues 2 to 11 and the carboxyl-terminal 70 residues. We demonstrated that whether SecAΔ11 or SecAΔ11/N95 was functional for promoting cell growth depended solely on the vivo level of the protein, which appeared to govern residual dimerization. All three SecA mutant proteins were defective for promoting cell growth unless they were highly overproduced. Cell fractionation revealed that SecAΔ11 and SecAΔ11/N95 were proficient in membrane association, although the formation of integral membrane SecA was reduced. The presence of a modestly higher level of SecAΔ11/N95 in the membrane and the ability of this protein to form dimers, as detected by chemical cross-linking, were consistent with the higher level of secA expression and better growth of the SecAΔ11/N95 mutant than of the SecAΔ11 mutant. Biochemical studies showed that SecAΔ11 and SecAΔ11/N95 had identical dimerization defects, while SecAΔ8 was intermediate between these proteins and wild-type SecA in terms of dimer formation. Furthermore, both SecAΔ11 and SecAΔ11/N95 were equally defective in translocation ATPase specific activity. Our studies showed that the nonessential carboxyl-terminal 70 residues of SecA play no role in its dimerization, while increasing the truncation of the amino-terminal region of SecA from 8 to 11 residues results in increased defects in SecA dimerization and poor in vivo function unless the protein is highly overexpressed. They also clarified a number of conflicting previous reports and support the essential nature of the SecA dimer.

scholarly journals The Hinge and Chromo Shadow Domain Impart Distinct Targeting of HP1-Like Proteins

2001 ◽  
Vol 21 (7) ◽  
pp. 2555-2569 ◽  
Author(s):  
James F. Smothers ◽  
Steven Henikoff
Keyword(s):  
Interphase Nuclei ◽  
Subnuclear Localization ◽  
Conserved Sequence ◽  
Amino Terminal ◽  
Sequence Variations ◽  
Specific Localization ◽  
Carboxyl Terminal ◽  
Chromo Shadow Domain ◽  
Hp1 Proteins

ABSTRACT Drosophila heterochromatin-associated protein 1 (HP1) is an abundant component of heterochromatin, a highly condensed compartment of the nucleus that comprises a major fraction of complex genomes. Some organisms have been shown to harbor multiple HP1-like proteins, each exhibiting spatially distinct localization patterns within interphase nuclei. We have characterized the subnuclear localization patterns of two newly discovered DrosophilaHP1-like proteins (HP1b and HP1c), comparing them with that of the originally described fly HP1 protein (here designated HP1a). While HP1a targets heterochromatin, HP1b localizes to both heterochromatin and euchromatin and HP1c is restricted exclusively to euchromatin. All HP1-like proteins contain an amino-terminal chromo domain, a connecting hinge, and a carboxyl-terminal chromo shadow domain. We expressed truncated and chimeric HP1 proteins in vivo to determine which of these segments might be responsible for heterochromatin-specific and euchromatin-specific localization. Both the HP1a hinge and chromo shadow domain independently target heterochromatin, while the HP1c chromo shadow domain is implicated solely in euchromatin localization. Comparative sequence analyses of HP1 homologs reveal a conserved sequence block within the hinge that contains an invariant sequence (KRK) and a nuclear localization motif. This block is not conserved in the HP1c hinge, possibly accounting for its failure to function as an independent targeting segment. We conclude that sequence variations within the hinge and shadow account for HP1 targeting distinctions. We propose that these targeting features allow different HP1 complexes to be distinctly sequestered in organisms that harbor multiple HP1-like proteins.

scholarly journals Ty3 Integrase Is Required for Initiation of Reverse Transcription

2002 ◽  
Vol 76 (6) ◽  
pp. 2804-2816 ◽  
Author(s):  
M. Henrietta Nymark-McMahon ◽  
Nadejda S. Beliakova-Bethell ◽  
Jean-Luc Darlix ◽  
Stuart F. J. Le Grice ◽  
Suzanne B. Sandmeyer
Keyword(s):  
Nuclear Localization ◽  
Reverse Transcription ◽  
Catalytic Triad ◽  
Catalytic Site ◽  
Nuclear Localization Sequence ◽  
Binding Motif ◽  
Site Mutation ◽  
Amino Terminal ◽  
Carboxyl Terminal

ABSTRACT The integrase (IN) encoded by the Saccharomyces cerevisiae retroviruslike element Ty3 has features found in retrovirus IN proteins including the catalytic triad, an amino-terminal zinc-binding motif, and a nuclear localization sequence. Mutations in the amino- and carboxyl-terminal domains of Ty3 IN cause reduced accumulation of full-length cDNA in the viruslike particles. We show that the reduction in cDNA is accompanied by reduced amounts of early intermediates such as minus-strand, strong-stop DNA. Expression of a capsid (CA)-IN fusion protein (CA-IN) complemented catalytic site and nuclear localization mutants, but not DNA mutants. However, expression of a fusion of CA, reverse transcriptase (RT), and IN (CA-RT-IN) complemented transposition of catalytic site and nuclear localization signal mutants, increased the amount of cDNA in some of the mutants, and complemented transposition of several mutants to low frequencies. Expression of a CA-RT-IN protein with a Ty3 IN catalytic site mutation did not complement transposition of either a Ty3 catalytic site mutant or a nuclear localization mutant but did increase the amount of cDNA in several mutants and complement at least one of the cDNA mutants for transposition. These in vivo data support a model in which independent IN domains can contribute to reverse transcription and integration. We conclude that during reverse transcription, the Ty3 IN domain interacts closely with the polymerase domain and may even constitute a domain within a heterodimeric RT. These studies also suggest that during integration the IN catalytic site and at least portions of the IN carboxyl-terminal domain act in cis.

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