scholarly journals Topological distribution of two connexin32 antigenic sites in intact and split rodent hepatocyte gap junctions.

1988 ◽  
Vol 107 (5) ◽  
pp. 1817-1824 ◽  
Author(s):  
D A Goodenough ◽  
D L Paul ◽  
L Jesaitis
Keyword(s):  
Amino Acids ◽  
Gap Junctions ◽  
Keyhole Limpet Hemocyanin ◽  
Cell Types ◽  
Cell Interaction ◽  
Amino Acid Sequences ◽  
Membrane Topology ◽  
Cdna Clones ◽  
Cleavage Sites ◽  
Mouse Hepatocyte

The membrane topology of connexin32, a principal polypeptide of gap junctions in diverse cell types, has been studied in rat and mouse hepatocyte gap junctions using site-specific antisera raised against synthetic oligopeptides corresponding to amino acid sequences deduced from cDNA clones. Based on published hydropathicity maps and identified protease-sensitive cleavage sites, oligopeptides were synthesized corresponding to two hydrophilic domains of connexin32, one predicted to face the cytoplasm, the other predicted to be directed extracellularly. Antisera were raised to keyhole limpet hemocyanin conjugates of the oligopeptides and used to map the distribution of their antigens using indirect immunocytochemistry on isolated gap junctions. The results directly demonstrated the cytoplasmic orientation of an antigen contained within amino acids 98-124 of the connexin32 sequence. The extracellular space in intact, isolated gap junctions is too small to permit binding of antibody molecules, necessitating the experimental separation of the junctional membranes to expose their extracellular surfaces using a urea/alkali procedure. While an antigen contained within amino acids 164-189 was visualized on the extracellular surfaces of some of the separated junctional membranes, variability in the observations and in the splitting procedure left ambiguities concerning the biological relevance of the observations after the denaturing conditions necessary to separate the junctional membranes. Using a different approach, however, the antigen could be exposed in intact liver using a hypertonic disaccharide junction-splitting procedure. The period of time of antigen exposure at the cell surface appears to peak at 30 s and disappear by 2-4 min. Taken together, these data demonstrate the extracellular orientation of an antigen contained within amino acids 164-189, which may be involved in cell-cell interaction within the gap junction.

Targeting motifs and functional parameters governing the assembly of connexins into gap junctions

2000 ◽  
Vol 349 (1) ◽  
pp. 281-287 ◽  
Author(s):  
Patricia E. M. MARTIN ◽  
James STEGGLES ◽  
Claire WILSON ◽  
Shoeb AHMAD ◽  
W. Howard EVANS
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gap Junctions ◽  
Gap Junction ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Lucifer Yellow ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Green Fluorescent

To study the assembly of gap junctions, connexin-green-fluorescent-protein (Cx-GFP) chimeras were expressed in COS-7 and HeLa cells. Cx26- and Cx32-GFP were targeted to gap junctions where they formed functional channels that transferred Lucifer Yellow. A series of Cx32-GFP chimeras, truncated from the C-terminal cytoplasmic tail, were studied to identify amino acid sequences governing targeting from intracellular assembly sites to the gap junction. Extensive truncation of Cx32 resulted in failure to integrate into membranes. Truncation of Cx32 to residue 207, corresponding to removal of most of the 78 amino acids on the cytoplasmic C-terminal tail, led to arrest in the endoplasmic reticulum and incomplete oligomerization. However, truncation to amino acid 219 did not impair Cx oligomerization and connexon hemichannels were targeted to the plasma membrane. It was concluded that a crucial gap-junction targeting sequence resides between amino acid residues 207 and 219 on the cytoplasmic C-terminal tail of Cx32. Studies of a Cx32E208K mutation identified this as one of the key amino acids dictating targeting to the gap junction, although oligomerization of this site-specific mutation into hexameric hemichannels was relatively unimpaired. The studies show that expression of these Cx-GFP constructs in mammalian cells allowed an analysis of amino acid residues involved in gap-junction assembly.

Cellular contacts between hindbrain and prospective ear during inductive interaction in the axolotl embryo

Development ◽  
1981 ◽  
Vol 66 (1) ◽  
pp. 27-41
Author(s):  
Pat G. Model ◽  
Lonny S. Jarrett ◽  
Robin Bonazzoli
Keyword(s):  
Structural Analysis ◽  
Gap Junctions ◽  
Surface Area ◽  
Cell Types ◽  
Cell Interaction ◽  
Ambystoma Mexicanum ◽  
Amphibian Embryo ◽  
Direct Communication ◽  
Inductive Interaction ◽  
Cell Processes

In the amphibian embryo, beginning in the late neurula and continuing through midtailbud stages, the developing medulla exerts an inductive influence on the prospective ear, effecting its determination. Fine structural analysis of the region of closest apposition between the two tissues in the axolotl (Ambystoma mexicanum) reveals that during this period, there is a significant increase in the surface area of the apposed cells through the projection of long finger-like processes that traverse the medulla-ear interspace and the appearance of many focal contacts between the two cell types. These contacts are small, varying in diameter from 10–30 nm and they exhibit the septilaminar appearance and 2–4 nm intercellular cleft characteristic of gap junctions. Once the ear has been determined, both the cell processes and the focal junctions between apposed cells virtually disappear. We suggest that the projection of processes from the surfaces of the apposed cells enhances the opportunity for cell interaction through the formation of very small gap junctions and that the junctions could provide the structural substrate for direct communication between medulla and ear during their inductive interaction.

scholarly journals Three novel brain tropomyosin isoforms are expressed from the rat alpha-tropomyosin gene through the use of alternative promoters and alternative RNA processing.

1990 ◽  
Vol 10 (4) ◽  
pp. 1729-1742 ◽  
Author(s):  
J P Lees-Miller ◽  
L O Goodwin ◽  
D M Helfman
Keyword(s):  
Amino Acids ◽  
Striated Muscle ◽  
Structural Characteristics ◽  
Single Gene ◽  
Transcription Unit ◽  
Amino Acid Sequences ◽  
Terminal Region ◽  
Cdna Clones ◽  
Alternative Promoters ◽  
Tropomyosin Isoforms

cDNA clones encoding three novel tropomyosins, termed TMBr-1, TMBr-2, and TMBr-3, were isolated and characterized from a rat brain cDNA library. All are derived from a single gene, which was previously found to express striated muscle alpha-tropomyosin and a number of other tropomyosin isoforms via an alternative splicing mechanism (N. Ruiz-Opazo and B. Nadal-Ginard, J. Biol. Chem. 262:4755-4765, 1987; D. F. Wieczorek, C. W. J. Smith, and B. Nadal-Ginard, Mol. Cell. Biol. 8:679-694, 1988). The derived amino acid sequences revealed that TMBr-1 contains 281 amino acids, TMBr-2 contains 251 amino acids, and TMBr-3 contains 245 amino acids. All three proteins contain a region that is identical to amino acids 81 through 258 of skeletal muscle alpha-tropomyosin. TMBr-1 is identical to striated muscle alpha-tropomyosin from amino acids 1 through 258 but contains a novel COOH-terminal region from amino acids 259 through 281. TMBr-2 and TMBr-3 both contain identical NH2-terminal sequences from amino acids 1 through 44 which were found to be expressed from a novel promoter. TMBr-3 contains the same COOH-terminal region as TMBr-1, whereas TMBr-2 contains a second novel COOH-terminal region. The genomic organization of the exons encoding TMBr-1, TMBr-2, and TMBr-3 were determined. These studies revealed a previously uncharacterized promoter located in the internal region of the alpha-TM gene as well as two novel COOH-terminal coding exons. The alpha-TM gene is a complex transcription unit containing 15 exons including two alternative promoters, two internal mutually exclusive exon cassettes, and four alternatively spliced 3' exons that encode four different COOH-terminal coding regions. A total of nine distinct mRNAs are known to be expressed from the alpha-TM gene in a cell type-specific manner in tissues such as striated muscle, smooth muscle, kidney, liver, brain, and fibroblasts. The mRNAs encoding TMBr-1, TMBr-2, and TMBr-3 were found to be expressed only in brain tissue, with TMBr-3 being expressed at much greater levels than TMBr-1 and TMBr-2. The individual structural characteristics of each brain alpha-tropomyosin isoform and their possible functions are discussed.

scholarly journals 088 Differential Expression of Expansin Genes Isolated from Sweet Cherry (Prunus avium L.) during Fruit Ripening

HortScience ◽  
2000 ◽  
Vol 35 (3) ◽  
pp. 403F-404
Author(s):  
Zhencai Wu ◽  
Paul A. Wiersma
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Fruit Development ◽  
Sweet Cherry ◽  
Prunus Avium ◽  
Expression Patterns ◽  
Amino Acid Sequences ◽  
Cdna Clones ◽  
Low Level ◽  
Expansin Gene

Expansins are a class of proteins that stimulate the extension of plant cell walls. Expansins have been found in nearly all growing plant tissues, such as hycopotyls, young seedlings, fibers, internodes, flower petals, and ripening fruits. We isolated two full-length expansin cDNA clones, Pruav-Exp1 and Pruav-Exp2, from sweet cherry (Prunus avium L.) fruit. Pruav-Exp1 has 1048 nucleotides encoding 254 amino acids, while Pruav-Exp2 has 1339 nucleotides encoding 250 amino acids. Deduced amino acid sequences of sweet cherry Pruav-Exp1 and Pruav-Exp2 share 72% identity. A Blast search of the GenBank database with the deduced amino acid sequences of Pruav-Exp1 and Pruav-Exp2 indicated a high sequence identity with other plant expansin genes. Interestingly, Pruav-Exp1 shares 99% identity of amino acid sequence with that of apricot expansin Pav-Exp1. Fragments from the 3' ends of Pruav-Exp1 and Pruav-Exp2 were cloned to generate gene-specific probes. These probes were used to study expansin gene expression in different tissues and during fruit development. Northern blot analysis showed different mRNA expression patterns for each gene. The mRNA of Pruav-Exp1 was expressed at the pink and ripe stages, but not at the early green and yellow stages of fruit development. The mRNA of Pruav-Exp2 was present earlier, from a low level in yellow expanding fruit, increasing to a high level at the pink stage and remaining at this level through the ripe stage. Both mRNAs were also expressed at a low level in flower, but not present in other tissues such as roots, leaves and peduncles. Our study indicates an expansin gene family is present in sweet cherry and suggests that two expansin genes may have different roles during fruit development and ripening.

Conservation between human and fungal squalene synthetases: similarities in structure, function, and regulation

1993 ◽  
Vol 13 (5) ◽  
pp. 2706-2717
Author(s):  
G W Robinson ◽  
Y H Tsay ◽  
B K Kienzle ◽  
C A Smith-Monroy ◽  
R W Bishop
Keyword(s):  
Human Fibroblasts ◽  
Control Point ◽  
Cell Types ◽  
Amino Acid Sequences ◽  
Sterol Synthesis ◽  
Cdna Clones ◽  
Yeast Saccharomyces Cerevisiae ◽  
Functional Conservation ◽  
Membrane Spanning ◽  
Squalene Synthetase

Squalene synthetase (farnesyl diphosphate:farnesyl diphosphate farnesyltransferase; EC 2.5.1.21) is thought to represent a major control point of isoprene and sterol biosynthesis in eukaryotes. We demonstrate structural and functional conservation between the enzymes from humans, a budding yeast (Saccharomyces cerevisiae), and a fission yeast (Schizosaccharomyces pombe). The amino acid sequences of the human and S. pombe proteins deduced from cloned cDNAs were compared to those of the known S. cerevisiae protein. All are predicted to encode C-terminal membrane-spanning proteins of approximately 50 kDa with similar hydropathy profiles. Extensive sequence conservation exists in regions of the enzyme proposed to interact with its prenyl substrates (i.e., two farnesyl diphosphate molecules). Many of the highly conserved regions are also present in phytoene and prephytoene diphosphate synthetases, enzymes which catalyze prenyl substrate condensation reactions analogous to that of squalene synthetase. Expression of cDNA clones encoding S. pombe or hybrid human-S. cerevisiae squalene synthetases reversed the ergosterol requirement of S. cerevisiae cells bearing ERG9 gene disruptions, showing that these enzymes can functionally replace the S. cerevisiae enzyme. Inhibition of sterol synthesis in S. cerevisiae and S. pombe cells or in cultured human fibroblasts by treatment with the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor lovastatin resulted in elevated levels of squalene synthetase mRNA in all three cell types.

scholarly journals Characterization and Construction of Functional cDNA Clones of Pariacoto Virus, the First Alphanodavirus Isolated outside Australasia

2000 ◽  
Vol 74 (11) ◽  
pp. 5123-5132 ◽  
Author(s):  
Karyn N. Johnson ◽  
Jean-Louis Zeddam ◽  
L. Andrew Ball
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Capsid Protein ◽  
Cultured Cells ◽  
Geographic Origin ◽  
Amino Acid Sequences ◽  
Cdna Clones ◽  
Homologous Proteins ◽  
Genomic Rnas ◽  
Rna Genome

ABSTRACT Pariacoto virus (PaV) was recently isolated in Peru from the Southern armyworm (Spodoptera eridania). PaV particles are isometric, nonenveloped, and about 30 nm in diameter. The virus has a bipartite RNA genome and a single major capsid protein with a molecular mass of 39.0 kDa, features that support its classification as aNodavirus. As such, PaV is the firstAlphanodavirus to have been isolated from outside Australasia. Here we report that PaV replicates in wax moth larvae and that PaV genomic RNAs replicate when transfected into cultured baby hamster kidney cells. The complete nucleotide sequences of both segments of the bipartite RNA genome were determined. The larger genome segment, RNA1, is 3,011 nucleotides long and contains a 973-amino-acid open reading frame (ORF) encoding protein A, the viral contribution to the RNA replicase. During replication, a 414-nucleotide long subgenomic RNA (RNA3) is synthesized which is coterminal with the 3′ end of RNA1. RNA3 contains a small ORF which could encode a protein of 90 amino acids similar to the B2 protein of other alphanodaviruses. RNA2 contains 1,311 nucleotides and encodes the 401 amino acids of the capsid protein precursor α. The amino acid sequences of the PaV capsid protein and the replicase subunit share 41 and 26% identity with homologous proteins of Flock house virus, the best characterized of the alphanodaviruses. These and other sequence comparisons indicate that PaV is evolutionarily the most distant of the alphanodaviruses described to date, consistent with its novel geographic origin. Although the PaV capsid precursor is cleaved into the two mature capsid proteins β and γ, the amino acid sequence at the cleavage site, which is Asn/Ala in all other alphanodaviruses, is Asn/Ser in PaV. To facilitate the investigation of PaV replication in cultured cells, we constructed plasmids that transcribed full-length PaV RNAs with authentic 5′ and 3′ termini. Transcription of these plasmids in cells recreated the replication of PaV RNA1 and RNA2, synthesis of subgenomic RNA3, and translation of viral proteins A and α.

scholarly journals Conservation between human and fungal squalene synthetases: similarities in structure, function, and regulation.

1993 ◽  
Vol 13 (5) ◽  
pp. 2706-2717 ◽  
Author(s):  
G W Robinson ◽  
Y H Tsay ◽  
B K Kienzle ◽  
C A Smith-Monroy ◽  
R W Bishop
Keyword(s):  
Human Fibroblasts ◽  
Control Point ◽  
Cell Types ◽  
Amino Acid Sequences ◽  
Sterol Synthesis ◽  
Cdna Clones ◽  
Yeast Saccharomyces Cerevisiae ◽  
Functional Conservation ◽  
Membrane Spanning ◽  
Squalene Synthetase

Squalene synthetase (farnesyl diphosphate:farnesyl diphosphate farnesyltransferase; EC 2.5.1.21) is thought to represent a major control point of isoprene and sterol biosynthesis in eukaryotes. We demonstrate structural and functional conservation between the enzymes from humans, a budding yeast (Saccharomyces cerevisiae), and a fission yeast (Schizosaccharomyces pombe). The amino acid sequences of the human and S. pombe proteins deduced from cloned cDNAs were compared to those of the known S. cerevisiae protein. All are predicted to encode C-terminal membrane-spanning proteins of approximately 50 kDa with similar hydropathy profiles. Extensive sequence conservation exists in regions of the enzyme proposed to interact with its prenyl substrates (i.e., two farnesyl diphosphate molecules). Many of the highly conserved regions are also present in phytoene and prephytoene diphosphate synthetases, enzymes which catalyze prenyl substrate condensation reactions analogous to that of squalene synthetase. Expression of cDNA clones encoding S. pombe or hybrid human-S. cerevisiae squalene synthetases reversed the ergosterol requirement of S. cerevisiae cells bearing ERG9 gene disruptions, showing that these enzymes can functionally replace the S. cerevisiae enzyme. Inhibition of sterol synthesis in S. cerevisiae and S. pombe cells or in cultured human fibroblasts by treatment with the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor lovastatin resulted in elevated levels of squalene synthetase mRNA in all three cell types.

Three novel brain tropomyosin isoforms are expressed from the rat alpha-tropomyosin gene through the use of alternative promoters and alternative RNA processing

1990 ◽  
Vol 10 (4) ◽  
pp. 1729-1742
Author(s):  
J P Lees-Miller ◽  
L O Goodwin ◽  
D M Helfman
Keyword(s):  
Amino Acids ◽  
Striated Muscle ◽  
Structural Characteristics ◽  
Single Gene ◽  
Transcription Unit ◽  
Amino Acid Sequences ◽  
Terminal Region ◽  
Cdna Clones ◽  
Alternative Promoters ◽  
Tropomyosin Isoforms

cDNA clones encoding three novel tropomyosins, termed TMBr-1, TMBr-2, and TMBr-3, were isolated and characterized from a rat brain cDNA library. All are derived from a single gene, which was previously found to express striated muscle alpha-tropomyosin and a number of other tropomyosin isoforms via an alternative splicing mechanism (N. Ruiz-Opazo and B. Nadal-Ginard, J. Biol. Chem. 262:4755-4765, 1987; D. F. Wieczorek, C. W. J. Smith, and B. Nadal-Ginard, Mol. Cell. Biol. 8:679-694, 1988). The derived amino acid sequences revealed that TMBr-1 contains 281 amino acids, TMBr-2 contains 251 amino acids, and TMBr-3 contains 245 amino acids. All three proteins contain a region that is identical to amino acids 81 through 258 of skeletal muscle alpha-tropomyosin. TMBr-1 is identical to striated muscle alpha-tropomyosin from amino acids 1 through 258 but contains a novel COOH-terminal region from amino acids 259 through 281. TMBr-2 and TMBr-3 both contain identical NH2-terminal sequences from amino acids 1 through 44 which were found to be expressed from a novel promoter. TMBr-3 contains the same COOH-terminal region as TMBr-1, whereas TMBr-2 contains a second novel COOH-terminal region. The genomic organization of the exons encoding TMBr-1, TMBr-2, and TMBr-3 were determined. These studies revealed a previously uncharacterized promoter located in the internal region of the alpha-TM gene as well as two novel COOH-terminal coding exons. The alpha-TM gene is a complex transcription unit containing 15 exons including two alternative promoters, two internal mutually exclusive exon cassettes, and four alternatively spliced 3' exons that encode four different COOH-terminal coding regions. A total of nine distinct mRNAs are known to be expressed from the alpha-TM gene in a cell type-specific manner in tissues such as striated muscle, smooth muscle, kidney, liver, brain, and fibroblasts. The mRNAs encoding TMBr-1, TMBr-2, and TMBr-3 were found to be expressed only in brain tissue, with TMBr-3 being expressed at much greater levels than TMBr-1 and TMBr-2. The individual structural characteristics of each brain alpha-tropomyosin isoform and their possible functions are discussed.

scholarly journals Combinations of two amino acids (Ala40 and Phe75 or Ser40 and Tyr75) in the coat protein of apple chlorotic leaf spot virus are crucial for infectivity

2007 ◽  
Vol 88 (9) ◽  
pp. 2611-2618 ◽  
Author(s):  
Hajime Yaegashi ◽  
Masamichi Isogai ◽  
Hiroko Tajima ◽  
Teruo Sano ◽  
Nobuyuki Yoshikawa
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Coat Protein ◽  
Leaf Spot ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Cdna Clones ◽  
Spot Virus ◽  
Chlorotic Leaf

Amino acid sequences of apple chlorotic leaf spot virus (ACLSV) coat protein (CP) were compared between 12 isolates from apple, plum and cherry, and 109 cDNA clones that were amplified directly from infected apple tissues. Phylogenetic analysis based on the amino acid sequences of CP showed that the isolates and cDNA clones were separated into two major clusters in which the combinations of the five amino acids at positions 40, 59, 75, 130 and 184 (Ala40-Val59-Phe75-Ser130-Met184 or Ser40-Leu59-Tyr75-Thr130-Leu184) were highly conserved within each cluster. Site-directed mutagenesis using an infectious cDNA clone of ACLSV indicated that the combinations of two amino acids (Ala40 and Phe75 or Ser40 and Tyr75) are necessary for infectivity to Chenopodium quinoa plants by mechanical inoculation. Moreover, an agroinoculation assay indicated that the substitution of a single amino acid (Ala40 to Ser40 or Phe75 to Tyr75) resulted in extreme reduction in the accumulation of viral genomic RNA, double-stranded RNAs and viral proteins (movement protein and CP) in infiltrated tissues, suggesting that the combinations of the two amino acids at positions 40 and 75 are important for effective replication in host plant cells.

scholarly journals Isolation and classification of a family of cyclin gene homologues in Lupinus luteus.

1997 ◽  
Vol 44 (1) ◽  
pp. 37-42 ◽  
Author(s):  
J Deckert ◽  
J Jeleńska ◽  
Z Zaborowska ◽  
A B Legocki
Keyword(s):  
Cell Cycle ◽  
Amino Acids ◽  
Phylogenetic Analysis ◽  
Amino Acid Sequences ◽  
Lupinus Luteus ◽  
Cdna Clones ◽  
Conserved Residues ◽  
The Press ◽  
Cyclin Gene

The lupine (Lupinus luteus cv. Ventus) cDNA clones encoding homologues of cyclin (CycB1;2, CycB1;3, CycB1;4) have been isolated from cDNA library prepared from roots inoculated with Bradyrhizobium lupini. Comparison of the deduced amino-acid sequences of CycB1;2, CycB1;3, CycB1;4 and previously described CycB1;1 (Deckert et al. 1996, Biochimie 78, 90-94) showed that they share 46-65% of identical amino acids. The presence of conserved residues (Renaudin et. al., in The Plant Cell Cycle, in the press; Renaudin et al., Plant Mol. Biol, in the press) along with phylogenetic analysis of known plant cyclins revealed that the four lupine sequences belong to subgroup 1 of B-like mitotic cyclins.

Sign in / Sign up

Export Citation Format

Share Document