Cloning and characterization of cDNAs encoding the ε-subunit of eukaryotic initiation factor-2B from rabbit and human

Agatha I. Asuru; Harry Mellor; N. Shaun B. Thomas; Lu Yu; Jane Jane Chen; John S. Crosby; Steven D. Hartson; Scot R. Kimball; Leonard S. Jefferson; Robert L. Matts

doi:10.1016/0167-4781(96)00054-1

Cloning and characterization of cDNAs encoding the ε-subunit of eukaryotic initiation factor-2B from rabbit and human

Agatha I. Asuru, Harry Mellor, N. Shaun B. Thomas, Lu Yu, Jane Jane Chen, John S. Crosby, Steven D. Hartson, Scot R. Kimball, Leonard S. Jefferson, Robert L. Matts

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Abstract

A rabbit reticulocyte lysate cDNA library was screened with a polyclonal antiserum directed against eukaryotic initiation factor eIF-2B (eIF-2B). A 2508 base pair cDNA (pA1) was isolated and determined to encode the E-subunit of eIF-2B based on the immunoreactivity of the fusion protein expressed from the cDNA in Escherichia coli and the presence of two peptide sequences obtained from two Vs fragments of purified nonrecombinant eIF-2Bε in the deduced amino acid sequence of the cDNA. The open reading frame of the cDNA began with the third nucleotide of the cDNA with the first AUG codon at nucleotide 522. Mutational analysis of pA1 indicated that the cDNA did not code for full-length eIF-2Bε. Seven missing codons of the reading-frame and the 71 nucleotide 5' non-coding region of the eIF-2Bε mRNA were obtained by 5' RACE. A human eIF-2Bε cDNA fragment, which corresponded to a similar 2.3 kb fragment generated by digestion of the rabbit pA1 cDNA with EcoRI, was isolated from a human histiocytic lymphoma (U-937) cell cDNA library constructed in λgt10. The nucleotide and amino acid sequences were highly conserved between the rabbit and human cDNAs, showing approx. 90% sequence identity within the open reading frame. Northern and Western blot analyses of reticulocyte lysate and other rabbit tissue extracts indicated that the eIF-2Bε polypeptide has a similar apparent molecular weight in all tissues examined, and is coded for by a single ~2.8 kilobase mRNA species which is ubiquitously expressed.

Original language	English (US)
Pages (from-to)	309-317
Number of pages	9
Journal	Biochimica et Biophysica Acta - Gene Structure and Expression
Volume	1307
Issue number	3
DOIs	https://doi.org/10.1016/0167-4781(96)00054-1
State	Published - Jul 17 1996

All Science Journal Classification (ASJC) codes

Structural Biology
Biophysics
Biochemistry
Genetics

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10.1016/0167-4781(96)00054-1

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Asuru, A. I., Mellor, H., Thomas, N. S. B., Yu, L., Chen, J. J., Crosby, J. S., Hartson, S. D., Kimball, S. R., Jefferson, L. S., & Matts, R. L. (1996). Cloning and characterization of cDNAs encoding the ε-subunit of eukaryotic initiation factor-2B from rabbit and human. Biochimica et Biophysica Acta - Gene Structure and Expression, 1307(3), 309-317. https://doi.org/10.1016/0167-4781(96)00054-1

@article{a928d7ea44ea41ada80aa4daeff89ac2,

title = "Cloning and characterization of cDNAs encoding the ε-subunit of eukaryotic initiation factor-2B from rabbit and human",

abstract = "A rabbit reticulocyte lysate cDNA library was screened with a polyclonal antiserum directed against eukaryotic initiation factor eIF-2B (eIF-2B). A 2508 base pair cDNA (pA1) was isolated and determined to encode the E-subunit of eIF-2B based on the immunoreactivity of the fusion protein expressed from the cDNA in Escherichia coli and the presence of two peptide sequences obtained from two Vs fragments of purified nonrecombinant eIF-2Bε in the deduced amino acid sequence of the cDNA. The open reading frame of the cDNA began with the third nucleotide of the cDNA with the first AUG codon at nucleotide 522. Mutational analysis of pA1 indicated that the cDNA did not code for full-length eIF-2Bε. Seven missing codons of the reading-frame and the 71 nucleotide 5' non-coding region of the eIF-2Bε mRNA were obtained by 5' RACE. A human eIF-2Bε cDNA fragment, which corresponded to a similar 2.3 kb fragment generated by digestion of the rabbit pA1 cDNA with EcoRI, was isolated from a human histiocytic lymphoma (U-937) cell cDNA library constructed in λgt10. The nucleotide and amino acid sequences were highly conserved between the rabbit and human cDNAs, showing approx. 90% sequence identity within the open reading frame. Northern and Western blot analyses of reticulocyte lysate and other rabbit tissue extracts indicated that the eIF-2Bε polypeptide has a similar apparent molecular weight in all tissues examined, and is coded for by a single ~2.8 kilobase mRNA species which is ubiquitously expressed.",

author = "Asuru, {Agatha I.} and Harry Mellor and Thomas, {N. Shaun B.} and Lu Yu and Chen, {Jane Jane} and Crosby, {John S.} and Hartson, {Steven D.} and Kimball, {Scot R.} and Jefferson, {Leonard S.} and Matts, {Robert L.}",

note = "Funding Information: We would like to thank Robin Hurst, Jiwen Sun and David Cooper for their technical assistance, Dr. Timothy Cox for his assistance in raising the eIF-2B antiserum, and Ron Brown and Kate Parkes for protein sequencing. Some of the equipment used to carry out this work was purchased with funds provided to Oklahoma State University by N.I.H. (BRSG grant RR07077). H.M. was supported by a grant from the Juvenile Diabetes Foundation (394124) and a training grant from the National Institute of Diabetes and Digestive and Kidney Diseases (DK07684). This work was supported in part by: N.I.H. grants from the National Institute of Environmental Health Sciences (ES-04299; R.L.M.), and the National Institute of Diabetes and Digestive and Kidney Diseases (DK-15658 and DK-13499, L.S.J.; DK16272, J.-J.C.); the Juvenile Diabetes Foundation (193147; S.R.K.); the Kay Kendall Leukaemia Fund (N.S.B.T.); the Oklahoma Center for the Advancement of Science and Technology (HRC-NIP-B004); and the Oklahoma Agricultural Experiment Station under Project Number 1975.",

year = "1996",

month = jul,

day = "17",

doi = "10.1016/0167-4781(96)00054-1",

language = "English (US)",

volume = "1307",

pages = "309--317",

journal = "Biochimica et Biophysica Acta - Gene Structure and Expression",

issn = "0167-4781",

publisher = "Elsevier B.V.",

number = "3",

}

Asuru, AI, Mellor, H, Thomas, NSB, Yu, L, Chen, JJ, Crosby, JS, Hartson, SD, Kimball, SR, Jefferson, LS & Matts, RL 1996, 'Cloning and characterization of cDNAs encoding the ε-subunit of eukaryotic initiation factor-2B from rabbit and human', Biochimica et Biophysica Acta - Gene Structure and Expression, vol. 1307, no. 3, pp. 309-317. https://doi.org/10.1016/0167-4781(96)00054-1

TY - JOUR

T1 - Cloning and characterization of cDNAs encoding the ε-subunit of eukaryotic initiation factor-2B from rabbit and human

AU - Asuru, Agatha I.

AU - Mellor, Harry

AU - Thomas, N. Shaun B.

AU - Yu, Lu

AU - Chen, Jane Jane

AU - Crosby, John S.

AU - Hartson, Steven D.

AU - Kimball, Scot R.

AU - Jefferson, Leonard S.

AU - Matts, Robert L.

N1 - Funding Information: We would like to thank Robin Hurst, Jiwen Sun and David Cooper for their technical assistance, Dr. Timothy Cox for his assistance in raising the eIF-2B antiserum, and Ron Brown and Kate Parkes for protein sequencing. Some of the equipment used to carry out this work was purchased with funds provided to Oklahoma State University by N.I.H. (BRSG grant RR07077). H.M. was supported by a grant from the Juvenile Diabetes Foundation (394124) and a training grant from the National Institute of Diabetes and Digestive and Kidney Diseases (DK07684). This work was supported in part by: N.I.H. grants from the National Institute of Environmental Health Sciences (ES-04299; R.L.M.), and the National Institute of Diabetes and Digestive and Kidney Diseases (DK-15658 and DK-13499, L.S.J.; DK16272, J.-J.C.); the Juvenile Diabetes Foundation (193147; S.R.K.); the Kay Kendall Leukaemia Fund (N.S.B.T.); the Oklahoma Center for the Advancement of Science and Technology (HRC-NIP-B004); and the Oklahoma Agricultural Experiment Station under Project Number 1975.

PY - 1996/7/17

Y1 - 1996/7/17

N2 - A rabbit reticulocyte lysate cDNA library was screened with a polyclonal antiserum directed against eukaryotic initiation factor eIF-2B (eIF-2B). A 2508 base pair cDNA (pA1) was isolated and determined to encode the E-subunit of eIF-2B based on the immunoreactivity of the fusion protein expressed from the cDNA in Escherichia coli and the presence of two peptide sequences obtained from two Vs fragments of purified nonrecombinant eIF-2Bε in the deduced amino acid sequence of the cDNA. The open reading frame of the cDNA began with the third nucleotide of the cDNA with the first AUG codon at nucleotide 522. Mutational analysis of pA1 indicated that the cDNA did not code for full-length eIF-2Bε. Seven missing codons of the reading-frame and the 71 nucleotide 5' non-coding region of the eIF-2Bε mRNA were obtained by 5' RACE. A human eIF-2Bε cDNA fragment, which corresponded to a similar 2.3 kb fragment generated by digestion of the rabbit pA1 cDNA with EcoRI, was isolated from a human histiocytic lymphoma (U-937) cell cDNA library constructed in λgt10. The nucleotide and amino acid sequences were highly conserved between the rabbit and human cDNAs, showing approx. 90% sequence identity within the open reading frame. Northern and Western blot analyses of reticulocyte lysate and other rabbit tissue extracts indicated that the eIF-2Bε polypeptide has a similar apparent molecular weight in all tissues examined, and is coded for by a single ~2.8 kilobase mRNA species which is ubiquitously expressed.

AB - A rabbit reticulocyte lysate cDNA library was screened with a polyclonal antiserum directed against eukaryotic initiation factor eIF-2B (eIF-2B). A 2508 base pair cDNA (pA1) was isolated and determined to encode the E-subunit of eIF-2B based on the immunoreactivity of the fusion protein expressed from the cDNA in Escherichia coli and the presence of two peptide sequences obtained from two Vs fragments of purified nonrecombinant eIF-2Bε in the deduced amino acid sequence of the cDNA. The open reading frame of the cDNA began with the third nucleotide of the cDNA with the first AUG codon at nucleotide 522. Mutational analysis of pA1 indicated that the cDNA did not code for full-length eIF-2Bε. Seven missing codons of the reading-frame and the 71 nucleotide 5' non-coding region of the eIF-2Bε mRNA were obtained by 5' RACE. A human eIF-2Bε cDNA fragment, which corresponded to a similar 2.3 kb fragment generated by digestion of the rabbit pA1 cDNA with EcoRI, was isolated from a human histiocytic lymphoma (U-937) cell cDNA library constructed in λgt10. The nucleotide and amino acid sequences were highly conserved between the rabbit and human cDNAs, showing approx. 90% sequence identity within the open reading frame. Northern and Western blot analyses of reticulocyte lysate and other rabbit tissue extracts indicated that the eIF-2Bε polypeptide has a similar apparent molecular weight in all tissues examined, and is coded for by a single ~2.8 kilobase mRNA species which is ubiquitously expressed.

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DO - 10.1016/0167-4781(96)00054-1

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SN - 0167-4781

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JO - Biochimica et Biophysica Acta - Gene Structure and Expression

JF - Biochimica et Biophysica Acta - Gene Structure and Expression

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ER -

Cloning and characterization of cDNAs encoding the ε-subunit of eukaryotic initiation factor-2B from rabbit and human

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