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Related Experiment Videos

Isolation of a cDNA coding for human galactosyltransferase.

H E Appert, T J Rutherford, G E Tarr

    Biochemical and Biophysical Research Communications
    |August 29, 1986
    PubMed
    Summary

    Researchers purified human milk galactosyltransferase and determined peptide sequences. They used these sequences to identify complementary DNA (cDNA) clones, successfully isolating the galactosyltransferase coding sequence from a human milk cDNA library.

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    Area of Science:

    • Biochemistry
    • Molecular Biology
    • Enzymology

    Background:

    • Human milk galactosyltransferase (EC 2.4.1.22) plays a crucial role in glycosylation.
    • Understanding its molecular structure is essential for further research.

    Purpose of the Study:

    • To purify human milk galactosyltransferase to homogeneity.
    • To obtain amino acid sequences of peptide fragments for molecular cloning.
    • To isolate and sequence the corresponding cDNA.

    Main Methods:

    • Affinity chromatography for enzyme purification.
    • Edman degradation for peptide sequencing.
    • Oligonucleotide probe construction and screening of a lambda gt10 cDNA library.
    • DNA sequencing of cDNA inserts.

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    Main Results:

    • Human milk galactosyltransferase was purified to homogeneity.
    • Amino acid sequences of eight peptide fragments were determined.
    • A 783 bp coding sequence for galactosyltransferase was identified within a 1.7 Kbp cDNA insert.
    • Two positive cDNA clones were isolated from 3 x 10^6 recombinants.

    Conclusions:

    • The study successfully purified human milk galactosyltransferase and identified its cDNA.
    • This provides a foundation for further studies on the enzyme's structure and function.