scholarly journals Phenotypic expression in Escherichia coli and nucleotide sequence of two Chinese hamster lung cell cDNAs encoding different dihydrofolate reductases.

1984 ◽  
Vol 4 (1) ◽  
pp. 38-48 ◽  
Author(s):  
P W Melera ◽  
J P Davide ◽  
C A Hession ◽  
K W Scotto
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Nucleotide Sequence ◽  
Dihydrofolate Reductase ◽  
Phenotypic Expression ◽  
Chinese Hamster ◽  
Differential Sensitivity ◽  
Nucleotide Sequence Analysis ◽  
Cdna Clones ◽  
Weight Class

Nucleotide sequence analysis of two cDNA clones, one shown to direct the synthesis in Escherichia coli of the pI 6.7 form of the 20,000-molecular-weight class of Chinese hamster lung cell dihydrofolate reductase, and the other shown to direct the synthesis of the pI 6.5 form of the 21,000-molecular-weight class of the enzyme, has revealed the following: (i) the differences in physical and enzymatic properties displayed by these two proteins are due to two variations in their respective amino acid sequences with the conversion of Leu to Phe at position 22 probably responsible for the differential sensitivity of these two enzymes to methotrexate and methasquin; (ii) the multiple mRNAs responsible for the synthesis of each of these proteins differ in size due, at least in part, to a length heterogeneity at their 3' ends; (iii) these two proteins are encoded by different genes; and (iv) the sequence AAATATA appears to be a major polyadenylation signal in one Chinese hamster lung cell dihydrofolate reductase gene and a minor signal in another.

Phenotypic expression in Escherichia coli and nucleotide sequence of two Chinese hamster lung cell cDNAs encoding different dihydrofolate reductases

1984 ◽  
Vol 4 (1) ◽  
pp. 38-48
Author(s):  
P W Melera ◽  
J P Davide ◽  
C A Hession ◽  
K W Scotto
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Nucleotide Sequence ◽  
Dihydrofolate Reductase ◽  
Phenotypic Expression ◽  
Chinese Hamster ◽  
Differential Sensitivity ◽  
Nucleotide Sequence Analysis ◽  
Cdna Clones ◽  
Weight Class

Nucleotide sequence analysis of two cDNA clones, one shown to direct the synthesis in Escherichia coli of the pI 6.7 form of the 20,000-molecular-weight class of Chinese hamster lung cell dihydrofolate reductase, and the other shown to direct the synthesis of the pI 6.5 form of the 21,000-molecular-weight class of the enzyme, has revealed the following: (i) the differences in physical and enzymatic properties displayed by these two proteins are due to two variations in their respective amino acid sequences with the conversion of Leu to Phe at position 22 probably responsible for the differential sensitivity of these two enzymes to methotrexate and methasquin; (ii) the multiple mRNAs responsible for the synthesis of each of these proteins differ in size due, at least in part, to a length heterogeneity at their 3' ends; (iii) these two proteins are encoded by different genes; and (iv) the sequence AAATATA appears to be a major polyadenylation signal in one Chinese hamster lung cell dihydrofolate reductase gene and a minor signal in another.

Construction and Identification by Partial Nucleotide Sequence Analysis of Bovine Casein and β-Lactoglobulin cDNA Clones

DNA ◽  
1982 ◽  
Vol 1 (4) ◽  
pp. 375-386 ◽  
Author(s):  
I.M. WILLIS ◽  
A.F. STEWART ◽  
A. CAPUTO ◽  
A.R. THOMPSON ◽  
A.G. MACKINLAY
Keyword(s):  
Sequence Analysis ◽  
Nucleotide Sequence ◽  
Nucleotide Sequence Analysis ◽  
Cdna Clones ◽  
Partial Nucleotide Sequence ◽  
Β Lactoglobulin

Mouse protamine 2 is synthesized as a precursor whereas mouse protamine 1 is not

1987 ◽  
Vol 7 (6) ◽  
pp. 2173-2179
Author(s):  
P C Yelick ◽  
R Balhorn ◽  
P A Johnson ◽  
M Corzett ◽  
J A Mazrimas ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Nucleotide Sequence ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Nucleotide Sequence Analysis ◽  
Cdna Clones ◽  
Protamine 1 ◽  
Protamine 2

The nuclei of mouse spermatozoa contain two protamine variants, mouse protamine 1 (mP1) and mouse protamine 2 (mP2). The amino acid sequence predicted from mP1 cDNAs demonstrates that mP1 is a 50-amino-acid protein with strong homology to other mammalian P1 protamines. Nucleotide sequence analysis of independently isolated, overlapping cDNA clones indicated that mP2 is initially synthesized as a precursor protein which is subsequently processed into the spermatozoan form of mP2. The existence of the mP2 precursor was confirmed by amino acid composition and sequence analysis of the largest of a set of four basic proteins isolated from late-step spermatids whose synthesis is coincident with that of mP1. The sequence of the first 10 amino acids of this protein, mP2 precursor 1, exactly matches that predicted from the nucleotide sequence of cDNA and genomic mP2 clones. The amino acid composition of isolated mP2 precursor 1 very closely matches that predicted from the mP2 cDNA nucleotide sequence. Sequence analysis of the amino terminus of isolated mature mP2 identified the final processing point within the mP2 precursor. These studies demonstrated that mP2 is synthesized as a precursor containing 106 amino acids which is processed into the mature, 63-amino-acid form found in spermatozoa.

scholarly journals Isolation, nucleotide sequence analysis, and disruption of the MDH2 gene from Saccharomyces cerevisiae: evidence for three isozymes of yeast malate dehydrogenase.

1991 ◽  
Vol 11 (1) ◽  
pp. 370-380 ◽  
Author(s):  
K I Minard ◽  
L McAlister-Henn
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Sequence Analysis ◽  
Nucleotide Sequence ◽  
Carbon Source ◽  
Malate Dehydrogenase ◽  
Sodium Dodecyl ◽  
Yeast Cells ◽  
Nucleotide Sequence Analysis ◽  
Haploid Strain

The major nonmitochondrial isozyme of malate dehydrogenase (MDH2) in Saccharomyces cerevisiae cells grown with acetate as a carbon source was purified and shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a subunit molecular weight of approximately 42,000. Enzyme assays and an antiserum prepared against the purified protein were used to screen a collection of acetate-nonutilizing (acetate-) yeast mutants, resulting in identification of mutants in one complementation group that lack active or immunoreactive MDH2. Transformation and complementation of the acetate- growth phenotype was used to isolate a plasmid carrying the MDH2 gene from a yeast genomic DNA library. The amino acid sequence derived from complete nucleotide sequence analysis of the isolated gene was found to be extremely similar (49% residue identity) to that of yeast mitochondrial malate dehydrogenase (molecular weight, 33,500) despite the difference in sizes of the two proteins. Disruption of the MDH2 gene in a haploid yeast strain produced a mutant unable to grow on minimal medium with acetate or ethanol as a carbon source. Disruption of the MDH2 gene in a haploid strain also containing a disruption in the chromosomal MDH1 gene encoding the mitochondrial isozyme produced a strain unable to grow with acetate but capable of growth on rich medium with glycerol as a carbon source. The detection of residual malate dehydrogenase activity in the latter strain confirmed the existence of at least three isozymes in yeast cells.

scholarly journals Nucleotide sequence analysis of the purEK operon encoding 5'-phosphoribosyl-5-aminoimidazole carboxylase of Escherichia coli K-12.

1989 ◽  
Vol 171 (1) ◽  
pp. 205-212 ◽  
Author(s):  
A A Tiedeman ◽  
J Keyhani ◽  
J Kamholz ◽  
H A Daum ◽  
J S Gots ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Sequence Analysis ◽  
Nucleotide Sequence ◽  
Nucleotide Sequence Analysis ◽  
K 12

scholarly journals Characteristics of a New Enantioselective Thermostable Dipeptidase from Brevibacillus borstelensis BCS-1 and Its Application to Synthesis of a d-Amino-Acid-Containing Dipeptide

2004 ◽  
Vol 70 (3) ◽  
pp. 1570-1575 ◽  
Author(s):  
Dae Heoun Baek ◽  
Jae Jun Song ◽  
Seok-Joon Kwon ◽  
Chung Park ◽  
Chang-Min Jung ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Open Reading Frame ◽  
Nucleotide Sequence Analysis ◽  
Reading Frame ◽  
E Coli ◽  
Specificity Constant ◽  
Optimal Ph

ABSTRACT A new thermostable dipeptidase gene was cloned from the thermophile Brevibacillus borstelensis BCS-1 by genetic complementation of the d-Glu auxotroph Escherichia coli WM335 on a plate containing d-Ala-d-Glu. Nucleotide sequence analysis revealed that the gene included an open reading frame coding for a 307-amino-acid sequence with an M r of 35,000. The deduced amino acid sequence of the dipeptidase exhibited 52% similarity with the dipeptidase from Listeria monocytogenes. The enzyme was purified to homogeneity from recombinant E. coli WM335 harboring the dipeptidase gene from B. borstelensis BCS-1. Investigation of the enantioselectivity (E) to the P1 and P1′ site of Ala-Ala revealed that the ratio of the specificity constant (k cat /Km ) for l-enantioselectivity to the P1 site of Ala-Ala was 23.4 � 2.2 [E = (k cat /Km ) l,d /(k cat /Km ) d,d ], while the d-enantioselectivity to the P1′ site of Ala-Ala was 16.4 � 0.5 [E = (k cat /Km ) l,d /(k cat /Km ) l,l ] at 55�C. The enzyme was stable up to 55�C, and the optimal pH and temperature were 8.5 and 65�C, respectively. The enzyme was able to hydrolyze l-Asp-d-Ala, l-Asp-d-AlaOMe, Z-d-Ala-d-AlaOBzl, and Z-l-Asp-d-AlaOBzl, yet it could not hydrolyze d-Ala-l-Asp, d-Ala-l-Ala, d-AlaNH2, and l-AlaNH2. The enzyme also exhibited β-lactamase activity similar to that of a human renal dipeptidase. The dipeptidase successfully synthesized the precursor of the dipeptide sweetener Z-l-Asp-d-AlaOBzl.

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