scholarly journals Import of the carboxy-terminal portion of acyl-CoA oxidase into peroxisomes of Candida tropicalis.

1987 ◽  
Vol 105 (1) ◽  
pp. 247-250 ◽  
Author(s):  
G M Small ◽  
P B Lazarow
Keyword(s):  
Cdna Clone ◽  
Candida Tropicalis ◽  
In Vitro Transcription ◽  
Major Product ◽  
Sufficient Information ◽  
Terminal Portion ◽  
Peroxisomal Protein ◽  
Acyl Coa Oxidase ◽  
Carboxy Terminal

We report the sequence of a cDNA clone that codes for the carboxy-terminal portion of the peroxisomal protein, acyl-CoA oxidase, from the yeast, Candida tropicalis. This is a newly identified acyl-CoA oxidase sequence, most likely a second allele of POX4. The cDNA clone was expressed by in vitro transcription followed by translation. The major product, a 43-kD protein, associated with isolated peroxisomes in an in vitro import assay. More than half of the peroxisome-associated protein was protected from added protease, implying that it was internalized within the organelle. These findings indicate that there is sufficient information in the carboxy-terminal portion of the protein to target it to peroxisomes.

Distinct domains of a nucleolar protein mediate protein kinase binding, interaction with nucleic acids and nucleolar localization

1998 ◽  
Vol 111 (17) ◽  
pp. 2615-2623 ◽  
Author(s):  
A. Das ◽  
J.H. Park ◽  
C.B. Hagen ◽  
M. Parsons
Keyword(s):  
Protein Kinase ◽  
Nucleic Acids ◽  
Protozoan Parasite ◽  
In Vitro Transcription ◽  
Nucleic Acid Binding ◽  
Binding Interaction ◽  
Amino Terminal ◽  
Amino Acid Region ◽  
Carboxy Terminal

Nopp44/46 is a phosphoprotein of the protozoan parasite Trypanosoma brucei that is localized to the nucleolus. Based on the primary sequence, Nopp44/46 appears to be a protein composed of distinct domains. This communication describes the relationship of these domains to the known functional interactions of the molecule and suggests that the amino-terminal region defines a novel homology region that functions in nucleolar targeting. We have previously shown that Nopp44/46 is capable of interacting with nucleic acids and associating with a protein kinase. Using in vitro transcription and translation, we now demonstrate that the nucleic acid binding function maps to the carboxy-terminal domain of the molecule, a region rich in arginine-glycine-glycine motifs. Our experiments reveal that a central region containing a high proportion of acidic residues is required for association with the protein kinase. Analysis of transfectants expressing epitope-tagged Nopp44/46 deletion constructs showed that the amino-terminal 96 amino acids allowed nuclear and nucleolar accumulation of the protein. This region of the molecule shows homology to several recently described nucleolar proteins. Deletion of a 27-amino-acid region within this domain abrogated nucleolar, but not nuclear, localization. These studies show that Nopp44/46 is composed of distinct modules, each of which plays a different role in molecular interactions. We suggest that this protein could facilitate interactions between sets of nucleolar molecules.

scholarly journals A highly conserved mouse gene with a propensity to form pseudogenes in mammals.

1988 ◽  
Vol 8 (7) ◽  
pp. 2797-2803 ◽  
Author(s):  
D L Heller ◽  
K M Gianola ◽  
L A Leinwand
Keyword(s):  
Dna Sequence ◽  
Cdna Clone ◽  
Restriction Analysis ◽  
In Vitro Transcription ◽  
Open Reading Frame ◽  
Cdna Clones ◽  
Reading Frame ◽  
Mouse Cdna ◽  
Processed Pseudogenes

A mouse cDNA clone corresponding to an abundantly transcribed poly(A)+ mRNA was found to be represented by 200 copies in mammalian genomes. To understand the origin and nature of this sequence family, we studied two genomic members and two cDNA clones from mouse liver. The DNA sequence of the coding strand of a full-length cDNA clone was shown to have an open reading frame capable of encoding a 25-kilodalton polypeptide that has not been previously described. In vitro transcription-translation experiments verified the presence of an open reading frame encoding a protein of the predicted size. Restriction analysis of genomic DNA and DNA sequence analysis of genomic clones indicated that many of the 200 members of this family represent processed pseudogenes, with one or a small number of active structural genes. The vast majority of the genomic copies are heterogeneous in length, truncated at their 5' ends with respect to the mRNA, and do not appear to have intervening sequences. Two distinct genomic members of this family were sequenced and found to represent incomplete copies of the mRNA. Both are 5' truncated at slightly different points with respect to the mRNA. Both pseudogenes have multiple base changes, insertions, and deletions relative to the mRNA, and one of them encodes the poly(A) tail of the mRNA. The expression of this gene family is highest in rapidly dividing cells such as early mouse embryos and testis, but was seen in all tissues tested. This gene shows extremely high sequence conservation, extending to chicken, amphibian, and nematode genomes. Surprisingly, the gene appears to exist in only one copy in these organisms.

A highly conserved mouse gene with a propensity to form pseudogenes in mammals

1988 ◽  
Vol 8 (7) ◽  
pp. 2797-2803
Author(s):  
D L Heller ◽  
K M Gianola ◽  
L A Leinwand
Keyword(s):  
Dna Sequence ◽  
Cdna Clone ◽  
Restriction Analysis ◽  
In Vitro Transcription ◽  
Open Reading Frame ◽  
Cdna Clones ◽  
Reading Frame ◽  
Mouse Cdna ◽  
Processed Pseudogenes

A mouse cDNA clone corresponding to an abundantly transcribed poly(A)+ mRNA was found to be represented by 200 copies in mammalian genomes. To understand the origin and nature of this sequence family, we studied two genomic members and two cDNA clones from mouse liver. The DNA sequence of the coding strand of a full-length cDNA clone was shown to have an open reading frame capable of encoding a 25-kilodalton polypeptide that has not been previously described. In vitro transcription-translation experiments verified the presence of an open reading frame encoding a protein of the predicted size. Restriction analysis of genomic DNA and DNA sequence analysis of genomic clones indicated that many of the 200 members of this family represent processed pseudogenes, with one or a small number of active structural genes. The vast majority of the genomic copies are heterogeneous in length, truncated at their 5' ends with respect to the mRNA, and do not appear to have intervening sequences. Two distinct genomic members of this family were sequenced and found to represent incomplete copies of the mRNA. Both are 5' truncated at slightly different points with respect to the mRNA. Both pseudogenes have multiple base changes, insertions, and deletions relative to the mRNA, and one of them encodes the poly(A) tail of the mRNA. The expression of this gene family is highest in rapidly dividing cells such as early mouse embryos and testis, but was seen in all tissues tested. This gene shows extremely high sequence conservation, extending to chicken, amphibian, and nematode genomes. Surprisingly, the gene appears to exist in only one copy in these organisms.

scholarly journals Studies of Nematode TFIIE Function Reveal a Link between Ser-5 Phosphorylation of RNA Polymerase II and the Transition from Transcription Initiation to Elongation

2001 ◽  
Vol 21 (1) ◽  
pp. 1-15 ◽  
Author(s):  
Seiji Yamamoto ◽  
Yoshinori Watanabe ◽  
Peter J. van der Spek ◽  
Tomomichi Watanabe ◽  
Hiroyuki Fujimoto ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
In Vitro Transcription ◽  
Pol Ii ◽  
Transcription System ◽  
Carboxy Terminal ◽  
Transition To Elongation ◽  
Ctd Phosphorylation

ABSTRACT The general transcription factor TFIIE plays important roles in transcription initiation and in the transition to elongation. However, little is known about its function during these steps. Here we demonstrate for the first time that TFIIH-mediated phosphorylation of RNA polymerase II (Pol II) is essential for the transition to elongation. This phosphorylation occurs at serine position 5 (Ser-5) of the carboxy-terminal domain (CTD) heptapeptide sequence of the largest subunit of Pol II. In a human in vitro transcription system with a supercoiled template, this process was studied using a human TFIIE (hTFIIE) homolog from Caenorhabditis elegans (ceTFIIEα and ceTFIIEβ). ceTFIIEβ could partially replace hTFIIEβ, whereas ceTFIIEα could not replace hTFIIEα. We present the studies of TFIIE binding to general transcription factors and the effects of subunit substitution on CTD phosphorylation. As a result, ceTFIIEα did not bind tightly to hTFIIEβ, and ceTFIIEβ showed a similar profile for binding to its human counterpart and supported an intermediate level of CTD phosphorylation. Using antibodies against phosphorylated serine at either Ser-2 or Ser-5 of the CTD, we found that ceTFIIEβ induced Ser-5 phosphorylation very little but induced Ser-2 phosphorylation normally, in contrast to wild-type hTFIIE, which induced phosphorylation at both Ser-2 and Ser-5. In transcription transition assays using a linear template, ceTFIIEβ was markedly defective in its ability to support the transition to elongation. These observations provide evidence of TFIIE involvement in the transition and suggest that Ser-5 phosphorylation is essential for Pol II to be in the processive elongation form.

The Ability of Fibrinogens, FI and FII, to Support ADP- Induced Platelet Aggregation

1983 ◽  
Vol 50 (02) ◽  
pp. 527-529 ◽  
Author(s):  
H M Phillips ◽  
A Mansouri ◽  
C A Perry
Keyword(s):  
Platelet Aggregation ◽  
Terminal Portion ◽  
Hepes Buffer ◽  
Carboxy Terminal

SummaryFibrinogen plays an integral part in ADP-induced platelet aggregation. Controversy exists in regard to the role of the carboxy termini of fibrinogen Aa chains in this reaction. We have attempted to clarify this problem in view of the availability of a highly purified FII fibrinogen fraction. Kabi fibrinogen or its purified fractions FI, FII and FIII-IV-V were added to washed platelets in the presence of Tyrode-HEPES buffer pH 7.4. Aggregation was initiated by the addition of calcium and ADP. These fibrinogen fractions equally promoted ADP-induced platelet aggregation. The major difference among these fractions is in their Aα chains. The FI fraction contains intact Aα chains while FII and FIH-IV-V fractions have one and two partially degraded Aα chains at the carboxy terminal portion respectively. We conclude that the carboxy terminal portion of the Aα chain does not play an important role in promoting ADP-induced platelet aggregation.

In vitro validation of a 3-dimensional transrectal ultrasound system for prostate biopsiess

2007 ◽  
Vol 30 (4) ◽  
pp. 77
Author(s):  
Derek Cool ◽  
Shi Sherebrin ◽  
Jonathan Izawa ◽  
Joseph Chin ◽  
Aaron Fenster
Keyword(s):  
Prostate Biopsy ◽  
3D Model ◽  
Transrectal Ultrasound ◽  
Surface Topology ◽  
Sufficient Information ◽  
Needle Guidance ◽  
3 Dimensional ◽  
High Incidence ◽  
3D Information

Introduction: Transrectal ultrasound (TRUS) prostate biopsy (Bx) is currently confined to 2D information to both target and record 3D Bx locations. Accurate placement of Bx needles cannot be verified without 3D information, and recording Bx sites in 2D does not provide sufficient information to accurately guide the high incidence of repeat Bx. We have designed a 3D TRUS prostate Bx system that augments the current 2D TRUS system and provides tools for biopsy-planning, needle guidance, and recording of the biopsy core locations entirely in 3D. Methods: Our Bx system displays a 3D model of the patient’s prostate, which is generated intra-procedure from a collection of 2D TRUS images, representative of the particular prostate shape. Bx targets are selected, needle guidance is facilitated, and 3D Bx sites are recorded within the 3D context of the prostate model. The complete 3D Bx system was validated, in vitro, by performing standard ten-core Bx on anatomical phantoms of two patient’s prostates. The accuracy of the needle-guidance, Bx location recording, and 3D model volume and surface topology were validated against a CT gold standard. Results: The Bx system successfully reconstructed the 3D patient prostate models with a mean volume error of 3.2 ± 7.6%. Using the 3D system, needles were accurately guided to the pre-determined targets with a mean error of 2.26 ± 1.03 mm and the 3D locations of the Bx cores were accurately recorded with a mean distance error of 1.47 ± 0.79 mm. Conclusion: We have successfully developed a 3D TRUS prostate biopsy system and validated the system in vitro. A pilot study has been initiated to apply the system clinically.

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