Nucleotide sequences of the cDNA and an intronless pseudogene for human lactate dehydrogenase-A isozyme

|February 15, 1985

Summary

Researchers identified the genetic blueprint for the human lactate dehydrogenase-A enzyme. They also discovered a non-functional copy of this gene, known as a pseudogene, which contains mutations preventing it from producing the protein. This study provides a detailed look at how these genetic sequences compare.

Area of Science:

Molecular genetics and Lactate dehydrogenase-A genomics
Human fibroblast gene expression analysis

Background:

No prior work had resolved the complete nucleotide sequence of the human lactate dehydrogenase-A isozyme. This gap motivated researchers to investigate the genetic architecture of this metabolic enzyme. Prior research has shown that lactate dehydrogenase-A plays a role in cellular energy production. However, the exact composition of its messenger ribonucleic acid remained unclear. That uncertainty drove the isolation of clones from human fibroblast libraries. Previous studies focused on protein-level analysis in other mammals. This project builds upon those foundations by providing the full genetic code. No prior work had fully characterized the associated non-functional genetic remnants in humans.

Purpose Of The Study:

The aim of this study was to determine the complete nucleotide sequence of the human lactate dehydrogenase-A isozyme. Researchers sought to characterize both the functional messenger ribonucleic acid and the associated non-functional genomic sequence. This project addressed the lack of detailed genetic information regarding this metabolic enzyme in humans. The team intended to identify structural differences between the active gene and its inactive counterpart. They also aimed to evaluate the evolutionary conservation of the protein by comparing it with pig sequences. This work clarifies the genetic basis of enzyme production in human fibroblasts. The researchers focused on identifying specific mutations that disable the pseudogene. This effort provides a comprehensive map of the genetic elements involved in lactate dehydrogenase-A expression.

Main Methods:

Review approach involved isolating eight specific clones from a human fibroblast library. Investigators characterized these sequences to ensure high fidelity. The team determined the complete nucleotide composition of the functional cDNA. They also isolated genomic clones to identify the non-functional genetic variant. The researchers performed detailed sequence alignments to compare the two genetic forms. Computational tools facilitated the identification of termination codons and deletions. This approach enabled the mapping of specific amino acid substitutions. The study relied on direct comparison to evaluate structural integrity.

Main Results:

Key findings from the literature show the complete human lactate dehydrogenase-A cDNA spans 1710 base pairs. This sequence includes a 999 base pair coding region. The researchers identified 92% homology between human and pig protein subunits. The pseudogene sequence spans 1635 base pairs. Comparison reveals 12.9% differences between the functional cDNA and the pseudogene. The pseudogene contains two termination codons and two deletions. Only four of 25 cytosine-guanine dinucleotides remain unchanged in the pseudogene. These structural alterations prevent the pseudogene from producing a functional enzyme.

Conclusions:

The researchers propose that the identified pseudogene lacks the ability to generate a functional protein. This conclusion stems from the presence of multiple premature termination signals within the sequence. Furthermore, the loss of specific arginine residues at the active site confirms its inactive status. The authors suggest that the high degree of sequence divergence between the functional gene and the pseudogene indicates a long evolutionary history. Synthesis and implications reveal that most cytosine-guanine dinucleotides have undergone significant modification. This observation supports theories regarding the mutational decay of non-coding genetic elements over time. The study highlights the structural differences that distinguish active genes from their disabled counterparts. These findings clarify the genetic landscape of human metabolic enzymes.

Keywords:

cDNA cloning genetic homology nucleotide sequence metabolic enzymes

Frequently Asked Questions

The researchers propose that the pseudogene is non-functional because it contains two premature termination codons, two deletions, and three critical arginine substitutions at the active site, which prevent the synthesis of a viable protein.

The study utilized a human fibroblast cDNA library to isolate eight clones, four of which provided the nearly complete 1710 base pair nucleotide sequence for the enzyme.

The researchers propose that the 1710 base pair sequence is necessary to encode the 999 base pair protein-coding region, alongside the 5' and 3' untranslated segments and the poly(dA) tail.

The authors compared the human cDNA sequence against the pseudogene to identify 114 transitions, 65 transversions, and 36 deletions or insertions, demonstrating a 12.9% divergence between the two sequences.

The researchers measured a 92% homology between the human LDH-A polypeptide and the pig LDH-A subunit, based on 27 amino acid differences out of 331 total residues.

The authors propose that the rapid loss of cytosine-guanine dinucleotides in the pseudogene, with only four of 25 remaining, reflects the accumulation of mutations following the gene's inactivation.

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