Steven S.-L. Li

Characterization of mouse ubiquitin‐like SMT3A and SMT3B cNDAS and gene/pseudogenes

Mouse SMT3A and SMT3B cDNAs encoding ubiquitin‐like proteins of 110 and 95 amino acids, respectively, were isolated and sequenced. The sequence of the first 92 amino acids (ending with the conserved Gly‐Gly) of mouse SMT3A exhibited two differences at amino acid no. 38 and 76 in comparison with that of human SMT3A. The C‐terminal 18 amino acid sequence of mouse SMT3A was completely different The nucleotide sequences have been deposited in the GenBank database (SMT3A cDNA, AF063847; SMT3A‐pg1 pseudogene, AF063846; SMT3B cDNA, L79948; SMT3B‐pg1, AF067824; SMT3B‐pg2, AF067825; SMT3B‐pg3, AF067826).

Molecular characterization of genetic mutation in human lactate dehydrogenase-A (M) deficiency.

Human lactate dehydrogenase-A mutant gene was isolated from the genomic DNA library of a patient deficient in LDH-A (Muscle) subunit. The nucleotide sequences of seven protein-coding exons were determined and a deletion of 20 base-pairs in exon 6 was found. This mutation results in a frame-shift translation and premature termination. The predicted incomplete LDH-A (M) subunit containing only 259 instead of 331 amino acids appears to be degraded rapidly, since no protein was detected immunologically (Maekawa et al., Am J Hum Genet 39:232-238, 1986). In addition, three synonymous (silent) substitutions, A to C, T to C, and G to A, were observed at codons 115, 160 and 172, respectively, in this LDH-A mutant gene.

Mapping of human lactate dehydrogenase-A, -B, and -C genes and their related sequences: the gene for LDHC is located with that for LDHA on chromosome 11

Human testis-specific lactate dehydrogenase-C (LDHC) gene-related sequences are located with the LDHA gene on chromosome 11. The LDHB gene is on chromosome 12. Chromosomes 1, 2, 4, 9, and 10 appear to contain LDHA gene-related sequences, whereas the X chromosome and chromosome 13 possess LDHB gene-related sequences.

CLONING AND DEVELOPMENTAL EXPRESSION OF XENOPUS CDNAS ENCODING THE ENHANCER OF SPLIT GROUCHO AND RELATED PROTEINS

The two full-length cDNAs encoding ESG1 (Enhancer of split groucho) and related AES (Amino Enhancer of split) proteins of 767 and 197 amino acids, respectively, were cloned and sequenced from the African frog Xenopus laevis. The amino acid sequence of Xenopus ESG1 protein had 61% identity to the full-length Drosophila groucho. Xenopus AES protein exhibited 91%, 58% and 48% identity to the mouse AES, amino-terminal regions of Xenopus ESG1 and Drosophila groucho, respectively. Northern blot analysis showed that widespread RNA expression of ESG1 of 2.8 kb, ESG2 of 3.6 kb and AES of 2.2 kb transcripts were seen in adult tissues, whereas ESG1 and AES transcripts of 2.8 kb and 2.2 kb, respectively, were ubiquitously expressed in the developing embryos. The overall structural relationships of ESG and AES proteins among human, mouse, rat, Xenopus and Drosophila were analysed.

Human testicular lactate dehydrogenase-C gene is interrupted by six introns at positions homologous to those of LDH-A (muscle) and LDH-B (heart) genes.

Human genomic clones containing parts of testis-specific lactate dehydrogenase-C gene of approximately 40 kilobases in length were isolated and characterized. The protein-coding sequence of human LDH-C gene is interrupted by six introns at positions homologous to those of mammalian LDH-A (muscle) and LDH-B (heart) genes, and exhibits 21%, 24% and 34% nucleotide differences with those of mouse LDH-C, human LDH-A and LDH-B genes, respectively.

Identification and characterization of the SMT3 cDNA and gene encoding ubiquitin-like protein from Drosophila melanogaster

A SMT3 cDNA encoding ubiquitin-like protein from Drosophila melanogaster was isolated and sequenced. Drosophila SMT3 genomic DNA was amplified by polymerase chain reaction, and its nucleotide sequence was found to be identical to that of the cDNA, indicating the absence of intron in its protein coding region. The sequence of 90 amino acids of Drosophila SMT3 exhibited 55%, 73%, 70% and 52% identity to yeast SMT3, human SMT3A, SMT3B and SMT3C protein.

Analysis of genetic mutations in human lactate dehydrogenase-A(M) deficiency using DNA conformation polymorphism in combination with polyacrylamide gradient gel and silver staining

Human lactate dehydrogenase (LDH)-A mutant gene was analyzed by polymerase chain reaction - DNA conformation polymorphism (DCP). We used polyacrylamide gradient gel and silver staining procedures for DCP analysis and observed abnormal migration patterns in individuals heterozygous for LDH-A deficiency. Further sequence determination of the mutant alleles consistently resulted in detection of base substitutions, a G to T transversion at codon 328 (GAG----TAG), and synonymous substitutions at codon 115, 160 and 172. Such mutations were easily detectable using the DCP technique. The DCP technique using the polyacrylamide gradient gel and silver staining method seems likely to be useful for the rapid screening of mutations and for further genotype detection.

Structural relatedness of mouse lactate dehydrogenase isozymes, A4 (muscle), B4 (heart), and C4 (testis).

Three homotetrameric lactate dehydrogenase isozymes, LDH-M(A4), LDH-H(B4), and LDH-X(C4), from DBA/2J mice have been purified by affinity chromatography. The amino acid compositions of the subunits A, B, and C, based on a molecular weight of 36,000, have been determined. The compositional relatedness of these isozymes indicates that subunits A (muscle) and B (heart) are more closely related to each other than to subunit C (testis). Tryptic peptide maps and amino acid compositions of some active site peptides appear to confirm the compositional relatedness among these isozymes. The sequence of the loop region of mouse C subunit seems to be markedly different from all known A and B sequences, and the structural and functional implications are discussed.

Detection and characterization of new genetic mutations in individuals heterozygous for lactate dehydrogenase-B(H) deficiency using DNA conformation polymorphism analysis and silver staining

Human lactate dehydrogenase (LDH) — B(H) mutant genes were analyzed by polymerase chain reaction (PCR) and DNA conformation polymorphism. We used polyacrylamide gradient gel and silver staining procedures for DCP analysis, and observed abnormal migration patterns in individuals heterozygous for the LDH-B deficiency. Subsequent sequence determination of the mutant alleles consistently resulted in detection of three single base substitutions (transversions), viz., a C to A at residue “35” (GCG, Ala→GAG, Glu), a T to G at residue “172” (TTT, Phe→GTT, Val), and an A to T at residue “176” (ATG, Met→TTG, Leu). Furthermore, mismatched PCR or amplification refractory mutation system was developed for the rapid screening and confirmation of these mutations. These amino acid replacements may cause conformational changes in neighboring residues; this probably affects the active site arrangement and results in the loss of enzyme activity.

Analysis of a genetic mutation in an electrophoretic variant of the human lactate dehydrogenase-B(H) subunit

An electrophoretic variant of the lactate dehydrogenase (LDH)-B(H) subunit was discovered in a patient with diabetes mellitus. His LDH activity in serum was slightly lower than normal and the LDH isozyme pattern showed an abnormal migration indicating an LDH-B subunit variant of the fast type. The LDH containing the variant subunit revealed a decreased heat stability. DNA analysis of the variant allele detected a base substitution, an A to G transition, at codon 6 (AAA→GAA). The mutation resulted in the replacement of a lysine by a glutamic acid (K6E). The change may cause the heat instability and affect the net charge of the variant subunit, resulting in an electrophoretic LDH-B subunit variant of the fast type.