Purification and structural characterization of ovine placental lactogen

1990 ◽  
Vol 126 (1) ◽  
pp. 141-149 ◽  
Author(s):  
W. C. Warren ◽  
R. Liang ◽  
G. G. Krivi ◽  
N. R. Siegel ◽  
R. V. Anthony
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Structural Characterization ◽  
Binding Sites ◽  
Untranslated Region ◽  
Fetal Liver ◽  
Radioreceptor Assay ◽  
Placental Lactogen ◽  
Predicted Amino Acid Sequence ◽  
Cdna Clones

ABSTRACT Discrepancies exist in the reported purity and biological activity of ovine placental lactogen (oPL), and little structural characterization has been reported. Ovine PL was purified from fetal cotyledonary tissue (day 100 of gestation) by monitoring activity with a recombinant bovine GH (bGH) liver radioreceptor assay. Two hundred grams of tissue yielded 4·2 mg of oPL, with an ∼ 1000-fold purification of oPL specific activity following initial tissue extraction. The oPL was radioiodinated and used in an ovine fetal liver (day 100 of gestation) radioreceptor assay to examine competitive displacement of oPL, ovine GH (oGH) and ovine prolactin (oPRL). The potency of oPL (ED50 = 0·18 nmol/l; ED50 is the quantity of ligand necessary to displace 50% of specifically bound 125I-labelled oPL) was greater than that of oGH (ED50 = 4·1 nmol/l) and oPRL (ED50 = 1·1 μmol/l) in competing for 125I-labelled oPL-binding sites. Attempts to sequence the NH2 terminus of oPL by vapour-phase sequencing indicated that the NH2 terminus was blocked. Purified oPL was subjected to trypsin and CnBr digestion, and two CnBr and six tryptic peptides were sequenced. The peptide sequences were compared with other PLs, oPRL and bGH for sequence similarity, and found to be most similar to bovine PL (bPL; 68% overall identity) and oPRL (47% overall identity). Complementary DNA (cDNA) clones were isolated for oPL by screening a λZAP cDNA library with a cDNA coding for bPL. Three cDNAs were nucleotide sequenced, and their combined sequence included 41 nucleotides of 5'-untranslated region, the complete coding region of pre-oPL (708 nucleotides) and a portion of the 3' untranslated region (158 nucleotides). The predicted amino acid sequence derived from the nucleotide sequence confirmed homology to bPL (67%) and oPRL (48%). Little amino acid sequence existed with other PLs (≤29%) or GH proteins (≤27%). These results suggest that oPL and oGH are more biologically similar in their ability to compete for fetal liver binding sites, but that oPL is structurally more similar to oPRL. Elucidation of exact structure–function relationships for oPL will, however, require further investigation. Journal of Endocrinology (1990) 126, 141–149

scholarly journals Cellular myosin heavy chain in human leukocytes: isolation of 5' cDNA clones, characterization of the protein, chromosomal localization, and upregulation during myeloid differentiation

Blood ◽  
1991 ◽  
Vol 78 (7) ◽  
pp. 1826-1833 ◽  
Author(s):  
LE Toothaker ◽  
DA Gonzalez ◽  
N Tung ◽  
RS Lemons ◽  
MM Le Beau ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Myosin Heavy Chain ◽  
Cell Lines ◽  
Heavy Chain ◽  
Myeloid Cell ◽  
Predicted Amino Acid Sequence ◽  
Cdna Clones ◽  
Granulocytic Differentiation ◽  
Human Leukocytes

Abstract We have isolated 5′ cDNA clones encoding a member of the cellular myosin heavy chain gene family from human leukocytes. The predicted amino acid sequence shows 93% identity to a chicken cellular myosin heavy chain, 76% to chicken smooth muscle, and 40% to human sarcomeric myosin heavy chain. The mRNA is expressed as a 7.4- to 7.9-kb doublet in many nonmuscle cells, and is upregulated in myeloid cell lines on induction from a proliferating to a differentiated state. Antisera raised against a peptide made from the predicted amino acid sequence specifically reacts with a 224-Kd polypeptide in leukocyte cell lines, and the protein is also upregulated during the induction of monocytic and granulocytic differentiation in these cells. The gene for this cellular myosin heavy chain maps to chromosome 22, bands q12.3-q13.1, demonstrating that it is not located in the previously described sarcomeric gene clusters on chromosomes 14 and 17. This cellular myosin heavy chain may be a major contractile protein responsible for movement in myeloid cell lines because no mRNA for sarcomeric myosin heavy chain is detected in these cells.

scholarly journals Amino acid sequence of band-3 protein from rainbow trout erythrocytes derived from cDNA

1992 ◽  
Vol 285 (1) ◽  
pp. 17-23 ◽  
Author(s):  
S Hübner ◽  
F Michel ◽  
V Rudloff ◽  
H Appelhans
Keyword(s):  
Rainbow Trout ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Untranslated Region ◽  
Band 3 ◽  
Cdna Clones ◽  
Amino Acid Residues ◽  
Band 3 Protein ◽  
Lower Vertebrate ◽  
Calculated Molecular Mass

In this report we present the first complete band-3 cDNA sequence of a poikilothermic lower vertebrate. The primary structure of the anion-exchange protein band 3 (AE1) from rainbow trout erythrocytes was determined by nucleotide sequencing of cDNA clones. The overlapping clones have a total length of 3827 bp with a 5′-terminal untranslated region of 150 bp, a 2754 bp open reading frame and a 3′-untranslated region of 924 bp. Band-3 protein from trout erythrocytes consists of 918 amino acid residues with a calculated molecular mass of 101 827 Da. Comparison of its amino acid sequence revealed a 60-65% identity within the transmembrane spanning sequence of band-3 proteins published so far. An additional insertion of 24 amino acid residues within the membrane-associated domain of trout band-3 protein was identified, which until now was thought to be a general feature only of mammalian band-3-related proteins.

scholarly journals Cellular myosin heavy chain in human leukocytes: isolation of 5' cDNA clones, characterization of the protein, chromosomal localization, and upregulation during myeloid differentiation

Blood ◽  
1991 ◽  
Vol 78 (7) ◽  
pp. 1826-1833
Author(s):  
LE Toothaker ◽  
DA Gonzalez ◽  
N Tung ◽  
RS Lemons ◽  
MM Le Beau ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Myosin Heavy Chain ◽  
Cell Lines ◽  
Heavy Chain ◽  
Myeloid Cell ◽  
Predicted Amino Acid Sequence ◽  
Cdna Clones ◽  
Granulocytic Differentiation ◽  
Human Leukocytes

We have isolated 5′ cDNA clones encoding a member of the cellular myosin heavy chain gene family from human leukocytes. The predicted amino acid sequence shows 93% identity to a chicken cellular myosin heavy chain, 76% to chicken smooth muscle, and 40% to human sarcomeric myosin heavy chain. The mRNA is expressed as a 7.4- to 7.9-kb doublet in many nonmuscle cells, and is upregulated in myeloid cell lines on induction from a proliferating to a differentiated state. Antisera raised against a peptide made from the predicted amino acid sequence specifically reacts with a 224-Kd polypeptide in leukocyte cell lines, and the protein is also upregulated during the induction of monocytic and granulocytic differentiation in these cells. The gene for this cellular myosin heavy chain maps to chromosome 22, bands q12.3-q13.1, demonstrating that it is not located in the previously described sarcomeric gene clusters on chromosomes 14 and 17. This cellular myosin heavy chain may be a major contractile protein responsible for movement in myeloid cell lines because no mRNA for sarcomeric myosin heavy chain is detected in these cells.

THE COMPLETE AMINO ACID SEQUENCE OF HUMAN FACTOR V

1987 ◽  
Author(s):  
Richard J Jenny ◽  
Debra D Pittman ◽  
John J Toole ◽  
Ronald W Kriz ◽  
Randal J Kaufman ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Factor Viii ◽  
Untranslated Region ◽  
Human Factor ◽  
Leader Peptide ◽  
Cdna Clones ◽  
Factor V ◽  
Human Factor Viii

cDNA clones encoding human factor V have been isolated and sequenced. The cDNA sequence of factor V obtained from overlapping clones includes a 6672 bp coding region, a 90 bp 5'-untranslated region and a 163 bp 3’-untranslated region including a poly-A tail. The deduced amino acid sequence consists of 2224 amino acids including a 28 amino acid leader peptide. A direct comparison to human factor VIII reveals considerable homology between both proteins with respect to amino acid sequence and domain structure. A triplicated "A" domain and duplicated "C" domain show an approximate 40% identity to the corresponding domains in factor VIII. Factor V and Factor VIII both possess a heavily glycosylated B domain that separates the heavy and light chains of the activated cofactors, although no significant homology is observed in this region. The B domain of factor V contains 35 tandem and approximately 9 additional semi - conserved repeats of nine amino acids of the form (D-L-S-Q-T-T-L-S-P) and 2 additional semi-conserved repeats of 17 amino acids. Factor V contains 37 potential N-linked glycosylation sites, 25 of which are in the B domain, and a total of 19 cysteine residues. By direct comparison to amino acid sequence obtained from both human and bovine factor V, the thrombin (IIa) cleavage sites have been assigned as Arg-709/Ser-710, Arg-1018/Thr-1019, and Are-1545/Ser-1546.(Supported by NIH Grant HL-34575)

scholarly journals The two beta-tubulin genes of Chlamydomonas reinhardtii code for identical proteins.

1984 ◽  
Vol 4 (12) ◽  
pp. 2686-2696 ◽  
Author(s):  
J Youngblom ◽  
J A Schloss ◽  
C D Silflow
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Chlamydomonas Reinhardtii ◽  
Yeast Cells ◽  
Predicted Amino Acid Sequence ◽  
Cdna Clones ◽  
Beta Tubulin ◽  
The Third ◽  
Tubulin Genes ◽  
Intervening Sequences

The two beta-tubulin genes of the unicellular green alga Chlamydomonas reinhardtii are expressed coordinately after deflagellation and produce two transcripts of 2.1 and 2.0 kilobases. Full-length cDNA clones corresponding to the transcript of each gene were isolated. DNA sequences were obtained from the cDNA clones and from cloned tubulin gene fragments. Both genes contained 1,332 base pairs of coding sequence, with only 19 nucleotide differences between the genes. Because all the differences occurred at the third base position of a codon and did not change the predicted amino acid sequence, we concluded that both beta-tubulin genes code for the same protein of 443 amino acids. The predicted amino acid sequence is 89 and 72% homologous with beta-tubulins from chicken and yeast cells, respectively. Each gene had three intervening sequences, which occurred at identical positions. Although the first two intervening sequences were not conserved between the two genes, the nucleotide sequence of the third intervening sequence was 89% conserved between the genes. The codon usage in the tubulin genes of C. reinhardtii was very biased: only 37 different codons were used. Striking differences occurred between the codons used in these nuclear genes and C. reinhardtii chloroplast genes.

cDNA cloning of transcription factor E4TF1 subunits with Ets and notch motifs

1993 ◽  
Vol 13 (3) ◽  
pp. 1385-1391
Author(s):  
H Watanabe ◽  
J Sawada ◽  
K Yano ◽  
K Yamaguchi ◽  
M Goto ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Amino Acid ◽  
Dna Binding ◽  
Amino Acid Sequence ◽  
Binding Activity ◽  
Amino Acid Sequences ◽  
Predicted Amino Acid Sequence ◽  
Cdna Clones ◽  
Dna Binding Activity

E4TF1 was originally identified as one of the transcription factors responsible for adenovirus E4 gene transcription. It is composed of two subunits, a DNA binding protein with a molecular mass of 60 kDa and a 53-kDa transcription-activating protein. Heterodimerization of these two subunits is essential for the protein to function as a transcription factor. In this study, we identified a new E4TF1 subunit, designated E4TF1-47, which has no DNA binding activity but can associate with E4TF1-60. We then cloned the cDNAs for each of the E4TF1 subunits. E4TF1 was purified, and the partial amino acid sequence of each subunit was determined. The predicted amino acid sequence of each cDNA clone revealed that E4TF1-60 had an ETS domain, which is a DNA binding domain common to ets-related transcription factors. E4TF1-53 had four tandemly repeated notch-ankyrin motifs. The putative cDNA of E4TF1-47 coded almost the same amino acid sequences as E4TF1-53. Three hundred and thirty-two amino acids of the N termini of E4TF1-47 and -53 were identical except for one amino acid insertion in E4TF1-53, and they differ from each other at the C terminus. These three recombinant cDNA clones were expressed in Escherichia coli, and the proteins behaved in the same manner as purified proteins in a gel retardation assay. Nucleotide and predicted amino acid sequences were highly homologous to GABP-alpha and -beta, which is further supported by the observation that GABP-specific antibody can recognize human E4TF1.

scholarly journals cDNA cloning of transcription factor E4TF1 subunits with Ets and notch motifs.

1993 ◽  
Vol 13 (3) ◽  
pp. 1385-1391 ◽  
Author(s):  
H Watanabe ◽  
J Sawada ◽  
K Yano ◽  
K Yamaguchi ◽  
M Goto ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Amino Acid ◽  
Dna Binding ◽  
Amino Acid Sequence ◽  
Binding Activity ◽  
Amino Acid Sequences ◽  
Predicted Amino Acid Sequence ◽  
Cdna Clones ◽  
Dna Binding Activity

E4TF1 was originally identified as one of the transcription factors responsible for adenovirus E4 gene transcription. It is composed of two subunits, a DNA binding protein with a molecular mass of 60 kDa and a 53-kDa transcription-activating protein. Heterodimerization of these two subunits is essential for the protein to function as a transcription factor. In this study, we identified a new E4TF1 subunit, designated E4TF1-47, which has no DNA binding activity but can associate with E4TF1-60. We then cloned the cDNAs for each of the E4TF1 subunits. E4TF1 was purified, and the partial amino acid sequence of each subunit was determined. The predicted amino acid sequence of each cDNA clone revealed that E4TF1-60 had an ETS domain, which is a DNA binding domain common to ets-related transcription factors. E4TF1-53 had four tandemly repeated notch-ankyrin motifs. The putative cDNA of E4TF1-47 coded almost the same amino acid sequences as E4TF1-53. Three hundred and thirty-two amino acids of the N termini of E4TF1-47 and -53 were identical except for one amino acid insertion in E4TF1-53, and they differ from each other at the C terminus. These three recombinant cDNA clones were expressed in Escherichia coli, and the proteins behaved in the same manner as purified proteins in a gel retardation assay. Nucleotide and predicted amino acid sequences were highly homologous to GABP-alpha and -beta, which is further supported by the observation that GABP-specific antibody can recognize human E4TF1.

scholarly journals Characterization of cDNA clones for human myeloperoxidase: Predicted amino acid sequence and evidence for multiple mRNA species

1987 ◽  
Vol 15 (5) ◽  
pp. 2013-2028 ◽  
Author(s):  
Keith R. Johnson ◽  
William M. Nauseef ◽  
Alessandra Care ◽  
Margaret J. Wheelock ◽  
Sara Shane ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Predicted Amino Acid Sequence ◽  
Cdna Clones ◽  
Mrna Species

scholarly journals The structure of human thrombospondin, an adhesive glycoprotein with multiple calcium-binding sites and homologies with several different proteins.

1986 ◽  
Vol 103 (5) ◽  
pp. 1635-1648 ◽  
Author(s):  
J Lawler ◽  
R O Hynes
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Binding Sites ◽  
Calcium Binding ◽  
Cdna Clones ◽  
Coding Region ◽  
Calcium Binding Sites ◽  
Mediate Cell ◽  
Type 3

Thrombospondin is one of a class of adhesive glycoproteins that mediate cell-to-cell and cell-to-matrix interactions. We have used two monoclonal antibodies to isolate cDNA clones of thrombospondin from a human endothelial cell cDNA library and have determined the complete nucleotide sequence of the coding region. Three regions of known amino acid sequence of human platelet thrombospondin confirm that the clones are authentic. Three types of repeating amino acid sequence are present in thrombospondin. The first is 57 amino acids long and shows homology with circumsporozoite protein from Plasmodium falciparum. The second is 50-60 amino acids long and shows homology with epidermal growth factor precursor. The third occurs as a continuous eightfold repeat of a 38-residue sequence; structural homology with parvalbumin and calmodulin indicates that these repeats constitute the multiple calcium-binding sites of thrombospondin. The amino acid sequence arg-gly-asp-ala is included in the last type 3 repeat. This sequence is probably the site for the association of thrombospondin with cells. In addition, localized homologies with procollagen, fibronectin, and von Willebrand factor are present in one region of the thrombospondin molecule.

The two beta-tubulin genes of Chlamydomonas reinhardtii code for identical proteins

1984 ◽  
Vol 4 (12) ◽  
pp. 2686-2696
Author(s):  
J Youngblom ◽  
J A Schloss ◽  
C D Silflow
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Chlamydomonas Reinhardtii ◽  
Yeast Cells ◽  
Predicted Amino Acid Sequence ◽  
Cdna Clones ◽  
Beta Tubulin ◽  
The Third ◽  
Tubulin Genes ◽  
Intervening Sequences

The two beta-tubulin genes of the unicellular green alga Chlamydomonas reinhardtii are expressed coordinately after deflagellation and produce two transcripts of 2.1 and 2.0 kilobases. Full-length cDNA clones corresponding to the transcript of each gene were isolated. DNA sequences were obtained from the cDNA clones and from cloned tubulin gene fragments. Both genes contained 1,332 base pairs of coding sequence, with only 19 nucleotide differences between the genes. Because all the differences occurred at the third base position of a codon and did not change the predicted amino acid sequence, we concluded that both beta-tubulin genes code for the same protein of 443 amino acids. The predicted amino acid sequence is 89 and 72% homologous with beta-tubulins from chicken and yeast cells, respectively. Each gene had three intervening sequences, which occurred at identical positions. Although the first two intervening sequences were not conserved between the two genes, the nucleotide sequence of the third intervening sequence was 89% conserved between the genes. The codon usage in the tubulin genes of C. reinhardtii was very biased: only 37 different codons were used. Striking differences occurred between the codons used in these nuclear genes and C. reinhardtii chloroplast genes.

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