Amino acid sequence diversity between bovine epidermal cytokeratin polypeptides of the basic (type II) subfamily as determined from cDNA clones

Two overlapping cDNA clones encoding pigeon liver carnitine acetyltransferase (EC 2.3.1.7) (CAT) were isolated from a pigeon liver lambda gt11 cDNA library by gene amplification using oligonucleotide primers based on the N-terminal amino acid sequence of the enzyme. The two clones, which represent the 5′ and 3′ ends of the gene, were spliced together to form a single cDNA construct containing the entire coding sequence for CAT, with an in-frame TGA stop codon 42 bases before the first ATG start site and a 3′-untranslated segment of 1057 bases. The largest open reading frame of 1942 nucleotides predicted a polypeptide of 627 amino acids and a molecular mass of 71.1 kDa. The N-terminus and four internal peptides from the amino acid sequence of pigeon breast muscle CAT were identified in the predicted sequence of the liver cDNA clone. The identity of the CAT cDNA was confirmed by heterologous expression of active recombinant CAT (rCAT) in insect cells using the baculovirus expression system. Western blots of rCAT from infected insect cell lysates and immunodetection with a rabbit anti-CAT polyclonal serum showed an immunoreactive protein band similar in size to native CAT from pigeon breast muscle. Like the native enzyme, rCAT was capable of acylating carnitine with a preference for small-chain acyl-CoAs of carbon chain lengths C2-C4.

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

Blood ◽

10.1182/blood.v78.7.1826.1826 ◽

1991 ◽

Vol 78 (7) ◽

pp. 1826-1833 ◽

Cited By ~ 45

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.

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Structural features of the low-molecular-mass human salivary mucin

Biochemical Journal ◽

10.1042/bj2870639 ◽

1992 ◽

Vol 287 (2) ◽

pp. 639-643 ◽

Cited By ~ 27

Author(s):

M S Reddy ◽

L A Bobek ◽

G G Haraszthy ◽

A R Biesbrock ◽

M J Levine

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Molecular Mass ◽

Gel Filtration ◽

Structural Features ◽

Cdna Expression Library ◽

Cdna Clones ◽

Expression Library ◽

Mrna Species ◽

Salivary Mucin

The low-molecular-mass human salivary mucin has at least two isoforms, MG2a and MG2b, that differ primarily in their sialic acid and fucose content. In this study, we characterize further these isoforms, particularly their peptide moieties. Trypsin digests of MG2a and MG2b yielded high- and low-molecular-mass glycopeptides following gel filtration on Sephacryl S-300. The larger glycopeptides from MG2a and MG2b had similar amino acid compositions and identical N-terminal sequences, suggesting common structural features between their peptides. An oligonucleotide probe generated from the amino acid sequence of the smaller glycopeptide from MG2a was employed in Northern-blot analysis. This probe specifically hybridized to two mRNA species from human submandibular and sublingual glands. A cDNA clone selected from a human submandibular gland cDNA expression library with antibody generated against deglycosylated MG2a also hybridized to these two mRNA species. In both cases, the larger mRNA was polydisperse, and the hybridization signal was more intense in the sublingual gland. In addition, the N-terminal amino acid sequence of the larger glycopeptide was found to be part of one of the selected MG2 cDNA clones.

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Cloning and sequencing of protochlorophyllide reductase

Biochemical Journal ◽

10.1042/bj2650789 ◽

1990 ◽

Vol 265 (3) ◽

pp. 789-798 ◽

Cited By ~ 46

Author(s):

P M Darrah ◽

S A Kay ◽

G R Teakle ◽

W T Griffiths

Keyword(s):

Amino Acid ◽

Chemical Analysis ◽

Amino Acid Sequence ◽

Cdna Library ◽

Avena Sativa ◽

Cdna Clones ◽

Lambda Phage ◽

Cloning And Sequencing ◽

Protochlorophyllide Reductase ◽

Cnbr Cleavage

Putative protochlorophyllide reductase cDNA clones (252 and 113) were isolated from an etiolated-oat (Avena sativa) cDNA library. These were used to indirectly characterize a further clone, p127, isolated from a lambda-phage gt11 cDNA library. The latter (1.15 kb in length) was sequenced, and the derived amino acid sequence was shown to be remarkably similar to that derived from chemical analysis of a CNBr-cleavage fragment of the purified reductase, p127 codes for more than 95% of the reductase protein.

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The amino acid sequence of component 7c, a type II intermediate-filament protein from wool

Biochemical Journal ◽

10.1042/bj2611015 ◽

1989 ◽

Vol 261 (3) ◽

pp. 1015-1022 ◽

Cited By ~ 26

Author(s):

L G Sparrow ◽

C P Robinson ◽

D T W McMahon ◽

M R Rubira

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Intermediate Filament ◽

Coiled Coil ◽

British Library ◽

Intermediate Filament Protein ◽

Type I ◽

Type Ii ◽

Blocking Group ◽

Intermediate Filament Proteins

Component 7c is one of the four homologous type II intermediate-filament proteins that, by association with the complementary type I proteins, form the microfibrils or intermediate filaments in wool. Component 7c was isolated as the S-carboxymethyl derivative from Merino wool and its amino acid sequence was determined by manual and automatic sequencing of peptides produced by chemical and enzymic cleavage reactions. It is an N-terminally blocked molecule of 491 residues and Mr (not including the blocking group) of 55,600; the nature of the blocking group has not been determined. The predicted secondary structure shows that component 7c conforms to the now accepted pattern for intermediate-filament proteins in having a central rod-like region of approximately 310 residues of coiled-coil alpha-helix flanked by non-helical N-and C-terminal regions. The central region is divided by three non-coiled-coil linking segments into four helical segments 1A, 1B, 2A and 2B. The N-and C-terminal non-helical segments are 109 and 71 residues respectively and are rich in cysteine. Details of procedures use in determining the sequence of component 7c have been deposited as a Supplementary Publication SUP 50152 (65 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1989) 257,5. The information comprises: (1) details of chemical and enzymic methods used for cleavage of component 7c, peptides CN1, CN2 and CN3, and various other peptides, (2) details of the procedures used for the fractionation and purification of peptides from (1), including Figures showing the elution profiles from the chromatographic steps used, (3) details of methods used to determine the C-terminal sequence of peptide CN3, and (4) detailed evidence to justify a number of corrections to the previously published sequence.

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