Structural organization and mapping of the human mitochondrial glycerol phosphate dehydrogenase-encoding gene and pseudogene.

Mitochondrial glycerol phosphate dehydrogenase (mtGPD) is the rate-limiting enzyme in the glycerol phosphate shuttle, which is thought to play an important role in cells that require an active glycolytic pathway. Abnormalities in mtGPD have been proposed as a potential cause for non-insulin-dependent diabetes mellitus. To facilitate genetic studies, we have isolated genomic clones containing the coding regions of the human mtGPD-encoding gene (GPDM). The gene contains 17 exons and is estimated to span more than 80 kb. All splice junctions contain GT/AG consensus sequences. Introns interrupt the sequences encoding the leader peptide, the FAD-binding site, the calcium-binding regions, and a conserved central element postulated to play a role in glycerol phosphate binding. Fluorescence in situ hybridization was used to map this gene to chromosome 2, band q24.1. A retropseudogene was identified and mapped to chromosome 17.

The sequence of the rat pyruvate carboxylase-encoding cDNA.

A composite 3945-bp cDNA that encodes rat pyruvate carboxylase (PC) has been constructed from clones isolated from a rat liver cell cDNA library and the nucleotide sequence has been determined. The rat cDNAs open reading frame encodes a protein of 1178 amino acids that is 98.6% identical (99.0% similar) to that of mouse PC and 96.0% identical (97.8% similar) to that of human PC.

Sequence of a cDNA encoding chicken vasoactive intestinal peptide (VIP).

Mammalian pre-pro-vasoactive intestinal peptide (pre-proVIP) gives rise to the neuropeptides vasoactive intestinal peptide (VIP) and peptide histidine isoleucine amide (PHI). The cDNA encoding chicken VIP was cloned and sequenced. The region of chicken pre-proVIP homologous to the mammalian PHI region is not followed by an amidation signal. This unusual feature suggests that processing of the precursor may be different in the chicken.

The sequence of a human mitochondrial glycerol-3-phosphate dehydrogenase-encoding cDNA.

A 2618-bp cDNA that encodes the human mitochondrial glycerol-3-phosphate dehydrogenase has been isolated from a HeLa cell cDNA library and the nucleotide sequence determined. An open reading frame encodes a protein of 727 amino acids that is 96% similar to the rat protein and, like the rat protein, contains sites homologous to the Ca(2+)-binding sites of calmodulin, as well as FAD- and putative glycerol-phosphate-binding sites.

The cooperative binding of chromosomal protein HMG-14 to nucleosome cores is reduced by single point mutations in the nucleosomal binding domain.

Mutants of human chromosomal protein HMG-14 were generated by site directed mutagenesis and used to study functional domains in this protein. A replacement of serine by cysteine at position 7 did not affect the binding of the protein to nucleosome cores. The sulfhydryl group in the nucleosome-bound protein is accessible to modifying agents suggesting that position 7 in the protein is not in close contact with either the DNA or the histones in the core particles. Under cooperative binding conditions, replacements of alanine by proline at position 21, or of lysine by cysteine at position 26, decreased the affinity of the protein for nucleosome cores 6.7- and 3-fold respectively. In contrast, the non-cooperative mode of binding was only minimally affected. A replacement of glutamic acid by glutamine at position 76 caused only minor changes in the binding of the protein to the cores. The results indicate that single point mutations, which change either the conformation or change in the nucleosomal binding domain of the protein, significantly reduce the ability of the HMG-14 protein to bind to nucleosome cores. We suggest that in chromatin the protein binds to nucleosomes in a cooperative manner and that upon binding to nucleosomes the protein acquires a distinct conformation.

Sequence of rat mitochondrial glycerol-3-phosphate dehydrogenase cDNA. Evidence for EF-hand calcium-binding domains.

The FAD-dependent, mitochondrial glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) is an essential component of the glycerol phosphate shuttle and is abundant in the pancreatic insulin cell, skeletal muscle, and brain. Although DNA clones for this enzyme and its homologues have been isolated from bacteria and yeast, it has never been cloned from a higher eukaryote. We have cloned and sequenced cDNAs encoding the rat mitochondrial glycerol-3-phosphate dehydrogenase. The longest cDNA (2337 base pairs) encodes a deduced protein of 727 amino acids that shows strong homology to the yeast and bacterial FAD-dependent glycerol phosphate dehydrogenases. The amino terminus of the purified mature protein was sequenced and shows identity with the deduced amino acid sequence beginning with residue 43. The 42 preceding amino acids are consistent with a mitochondrial leader peptide. A highly conserved FAD-binding domain and conserved regions possibly involved with glycerol phosphate binding are present. An unexpected finding was the homology of the deduced protein to calmodulin. Analysis of the deduced protein sequence shows a region near the carboxyl terminus containing two sequences homologous to "EF-hand" calcium-binding domains that are not present in the shorter yeast and bacterial homologues. The second of these domains appears to have features compatible with considerable affinity for calcium, whereas the first does not. The finding of a potential calcium-binding region is consistent with the known enhancement by calcium of the mammalian enzyme activity at low substrate concentrations and the lack of a requirement for calmodulin. This is the first report of EF-hands in a metabolic enzyme or in a mitochondrial protein.

Evolutionarily conserved motifs and protein binding elements in the 5' region of the chromosomal protein HMG-14 gene.

Although the structure of several genes coding for chromosomal proteins HMG-14 and HMG-17 has been determined, the mechanisms regulating the expression of these genes has not yet been examined. Toward this goal, we have cloned and sequenced a fragment containing the first three exons and 956 bp upstream from the start of translation of the functional mouse HMG-14 gene. Comparison of this sequence to the known sequence of the human HMG-14 gene revealed the presence of five distinct blocks of high sequence identity flanking the start of transcription and the CAAT box. DNase I and mobility-shift analysis identified a DNA region, downstream from the start of transcription, which may be important for the formation of a stable protein-DNA complex. Affinity chromatography on columns containing oligonucleotides corresponding to this sequence indicates that this region is a protein binding site.

A general approach to testing multifaceted personality constructs.

Some personality characteristics are composed of multiple, distinct subcomponents (e.g., Type A, hardiness, attributional style, self-monitoring). The advantages and disadvantages of 3 typical approaches to testing these constructs are reviewed. An alternative approach based on structural equation modeling is then offered. This approach has many advantages over its alternatives, including the provision of an explicit test of the structure of the multifaceted construct, the simultaneous test of the effects of this general construct and the unique aspects of its subcomponents, and the explicit consideration of measurement error. Although the modeling approach does have limitations, these limitations are equally applicable to all of its alternatives. Indeed, the principal disadvantages of the modeling approach seem to be its statistical complexity and the lack of education regarding its proper use.

Recombinant human chromosomal proteins HMG-14 and HMG-17.

Vectors for expressing human chromosomal proteins HMG-14 and HMG-17 in bacterial cultures under the control of the temperature-inducible lambda PL promoter have been constructed. The open reading frames of the cDNAs have been amplified by the polymerase chain reaction (PCR), utilizing amplimers containing desired restriction sites, thereby facilitating precise location of the initiation codon downstream from a ribosomal binding site. Expression of the recombinant proteins does not significantly affect bacterial growth. The rate of synthesis of the recombinant proteins is maximal during the initial stages of induction and slows down appreciably with time. After an initial burst of protein synthesis, the level of the recombinant protein in the bacterial extracts remains constant at different times following induction. Methods for rapid extraction and purification of the recombinant proteins are described. The recombinant proteins are compared to the proteins isolated from eucaryotic cells by electrophoretic mobility, Western analysis and nucleosome core mobility-shift assays. The ability of the proteins to shift the mobility of the nucleosome cores, but not that of DNA, can be used as a functional assay for these HMG proteins. A source for large quantities of human chromosomal proteins HMG-14 and HMG-17 will facilitate studies on their structure, cellular function and mechanism of interaction with nucleosomes.

Alternative processing of mRNAs encoding mammalian chromosomal high-mobility-group proteins HMG-I and HMG-Y.

The high-mobility-group protein HMG-I is a well-characterized nonhistone chromosomal protein that is preferentially expressed in rapidly dividing cells, binds to A. T-rich regions of DNA in vitro, and has been localized to particular regions of mammalian metaphase chromosomes. We isolated eight cDNA clones encoding HMG-I and its isoform HMG-Y from a human Raji cell cDNA library and detected blocks of nucleotide sequence rearrangements in the 5'-untranslated regions of these clones. In addition to this leader sequence variation, five of the eight cDNA clones had either a 33- or 36-base-pair in-frame deletion in their open reading frame (ORF); we found that this shortened ORF encodes the HMG-Y protein isoform. We present evidence that the 5'-untranslated-region and ORF heterogeneity of the cDNA clones is the result of alternative processing of RNA transcripts from a single functional gene. Several additional but probably nonfunctional HMG-I or HMG-Y gene copies exist in the human genome; we isolated and partially sequenced one of these pseudogenes and found that it is a processed HMG-Y retropseudogene.

Complete murine cDNA sequence, genomic structure, and tissue expression of the high mobility group protein HMG-I(Y).

A cDNA coding for the non-histone chromosomal protein HMG-I, or its isoform HMG-Y, was isolated from a murine Friend cell library using synthetic oligonucleotide hybridization probes. Sequence analysis showed that the 1670-base pair full length cDNA insert consists of a 201-base pair, G/C-rich (74%), 5'-untranslated region, a 288-base pair amino acid coding sequence, and an unusually long 1182-base pair 3'-untranslated region. The deduced 96-residue amino acid coding sequence of the murine HMG-I(Y) cDNA is very similar to the reported amino acid sequence of human HMG-I, except that it lacks 11 internal amino acids reported in the human protein. Based on Southern blot hybridization analysis of genomic DNA, there appear to be fewer than five copies of HMG-I(Y) genes in the haploid murine genome. These murine HMG-I(Y) genes contain a large (at least 890 base pairs) exon that includes most, or all, of the 3'-untranslated region; whereas the much shorter 5'-untranslated region and amino acid coding sequences are interrupted by at least one intron. A single size class (approximately 1700 nucleotides in murine cells and 2000 nucleotides in human cells) of HMG-I(Y) mRNAs was detected at high levels in total RNA extracts from rapidly dividing, transformed cells, but to a lesser extent, or not at all, in extracts from slowly or non-dividing cells.

A conformational study of the sequence specific binding of HMG-I (Y) with the bovine interleukin-2 cDNA.

The DNA sequence specific interaction of the high mobility group non-histone protein HMG-I (Y) with the 3' untranslated region of the bovine interleukin-2 cDNA has been studied. Circular dichroism and thermal denaturation studies suggest that HMG-I (Y) alters the conformational state and increases the thermal stability of the DNA. Additionally, amino acid sequence analysis suggests that the previously identified non-histone protein HMG-Y is an isoform of HMG-I.