Hypoxically inducible barley lactate dehydrogenase: cDNA cloning and molecular analysis

Journal Article · Sat Sep 01 00:00:00 EDT 1990 · Proceedings of the National Academy of Sciences of the United States of America; (United States)

DOI:https://doi.org/10.1073/pnas.87.18.7300· OSTI ID:5394563

Hondred, D ^[1]; Hanson, A D ^[2]

---

1. Michigan State Univ., East Lansing (USA)
2. Michigan State Univ., East Lansing (USA) Univ. de Montreal, Quebec (Canada)

In the roots of barley and other cereals, hypoxia induces a set of five isozymes of L-lactate dehydrogenase (LDH; (S)-lactate:NADH oxidoreductase, EC 1.1.1.27). Biochemical and genetic data indicate that the five LDH isozymes are tetramers that arise from random association of the products of two Ldh loci. To investigate this system, cDNA clones of LDH were isolated from a {lambda}gt11 cDNA library derived from hypoxically treated barley roots. The library was screened with antiserum raised against barley LDH purified {approx}3,000-fold by an improved three-step procedure. Immunopositive clones were rescreened with a cDNA probe synthesized by the polymerase chain reaction using primers modeled from the amino acid sequences of two tryptic LDH peptides. Two types of LDH clones were found. Nucleotide sequence analysis of one representative insert of each type (respectively, 1,305 and 1,166 base pairs) revealed open reading framed encoding 10 peptide fragments of LDH. The 1,305-base-pair insert included the entire coding region of a 356-residue LDH monomer. The nucleotide sequences of the two LDH cDNAs were 92% identical in the coding region, but highly divergent in the 3{prime} noncoding region, and thus probably correspond to the two postulated Ldh loci. The deduced amino acid sequences of the two barley LDHs were 96% identical to each other and very similar to those from vertebrate and bacterial LDHs. RNA blot hybridization showed a single mRNA band of 1.5 kilobases whose level rose about 8-fold in roots during hypoxic induction, as did the level of translatable LDH message.

• DOE Contract Number: AC02-76ER01338
• OSTI ID: 5394563
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (United States), Vol. 87:18; ISSN 0027-8424
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
LACTATE DEHYDROGENASE
MOLECULAR BIOLOGY
RECOMBINANT DNA
DNA-CLONING
AMINO ACID SEQUENCE
BARLEY
DNA POLYMERASES
DNA SEQUENCING
ELECTROPHORESIS
GENE REGULATION
ISOENZYMES
MAN
MESSENGER-RNA
PURIFICATION
ROOTS
ANIMALS
CEREALS
CLONING
DNA
DNA HYBRIDIZATION
ENZYMES
GRAMINEAE
HEMIACETAL DEHYDROGENASES
HYBRIDIZATION
LILIOPSIDA
MAGNOLIOPHYTA
MAMMALS
MOLECULAR STRUCTURE
NUCLEIC ACIDS
NUCLEOTIDYLTRANSFERASES
ORGANIC COMPOUNDS
OXIDOREDUCTASES
PHOSPHORUS-GROUP TRANSFERASES
PLANTS
POLYMERASES
PRIMATES
RNA
STRUCTURAL CHEMICAL ANALYSIS
TRANSFERASES
VERTEBRATES
550200* - Biochemistry