Genomic sequence comparison of the human and mouse adenosine deaminase gene regions

A challenge for mammalian genetics is the recognition of critical regulatory regions in primary gene sequence. One approach to this problem is to compare sequences from genes exhibiting highly conserved expression patterns in disparate organisms. Previous transgenic and transfection analyses defined conserved regulatory domains in the mouse and human adenosine deaminase (ADA) genes. We have thus attempted to identify regions with comparable similarity levels potentially indicative of critical ADA regulatory regions. On the basis of aligned regions of the mouse and human ADA gene, using a 24-bp window, we find that similarity overall (67.7%) and throughout the noncoding sequences (67.1%) is markedly lower than that of the coding regions (81%). This low overall similarity facilitated recognition of more highly conserved regions. In addition to the highly conserved exons, ten noncoding regions >100 bp in length displayed >70% sequence similarity. Most of these contained numerous 24-bp windows with much higher levels of similarity. A number of these regions, including the promoter and the thymic enhancer, were more similar than several exons. A third block, located near the thymic enhancer but just outside of a minimally defined locus control region, exhibited stronger similarity than the promoter or thymic enhancer. In contrast, only fragmentary similarity was exhibited in a region that harbors a strong duodenal enhancer in the human gene. These studies show that comparative sequence analysis can be a powerful tool for identifying conserved regulatory domains, but that some conserved sequences may not be detected by certain functional analyses as transgenic mice. Received: 27 March 1998 / Accepted: 22 September 1998     

Comparative sequence analysis of imprinted genes between human and mouse to reveal imprinting signatures

We performed a comparative genomic sequence analysis between human and mouse for 24 imprinted genes on human chromosomes 1, 6, 7, 11, 13, 14, 15, 18, 19, and 20. The MEME program was used to search for motifs within conserved sequences among the imprinted genes and we then used the MAST program to analyze for the presence or absence of motifs in the imprinted genes and 128 nonimprinted genes. Our analysis identified 15 motifs that were significantly enriched in the imprinted genes. We generated a logistic regression model by combining multiple motifs as input variables and the 24 imprinted genes and the 128 nonimprinted genes as a training set. The accuracy, sensitivity, and specificity of our model were 98, 92, and 99%, respectively. The model was further validated by an open test on 12 additional imprinted genes. The motifs identified in this study are novel imprinting signatures, which should improve our understanding of genomic imprinting and the role of genomic imprinting in human diseases.     

The chicken vimentin gene. Nucleotide sequence, regulatory elements, and comparison to the hamster gene

Here we report the nucleotide sequence of the chicken vimentin gene and its deduced primary amino acid sequence. A comparison of this gene to other intermediate filament protein genes demonstrates that both exon size and position are strongly conserved features of this multigene family. In addition, the hamster and chicken vimentin genes exhibit strong identity at the level of nucleotide (74%) and amino acid (80%) sequence. Interestingly, 40% of total sequence diversity is localized to the N terminus or "head" region of these genes whereas other protein domains (rod and C terminus) are remarkably identical in both nucleotide (81%) and amino acid (89%) sequence. Even stronger amino acid identity (100%) is exhibited in certain subdomains which may define regions crucial for filament formation and function. Not surprisingly, vimentin is more homologous across animal species than it is to other intermediate filament protein members (e.g. desmin) within the same species. A comparison of 5'-flanking sequences of the hamster and chicken genes as well as other characterized promoter elements (SV40, HSV-TK) reveals homologous sequence elements which may define common and/or unique sites involved in the modulation of gene expression. The implications of these sequence elements for both tissue-specific and developmental expression of the vimentin gene are discussed.     

Cloning and sequence analysis of the human liver rhodanese: comparison with the bovine and chicken enzymes

The cDNA for the human rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1), a nuclearly encoded protein of the mitochondrial matrix, was isolated from a human fetal liver cDNA library. Nucleotide sequence revealed an open reading frame coding for a polypeptide of 295 amino acids, which presented a 57% and 58% identity with the bovine and avian rhodanese, respectively. The analysis of the 5'-ends of the coding region gave no evidence for the presence of a cleavable signal sequence as found in other mitochondrial proteins. A comparison with two available amino acid sequences (cow and chicken) showed that sequence similarity is not restricted to the alpha-helices and beta-structures motifs which are remarkably superimposable in the two halves of bovine rhodanese, but extends to adjacent regions.     

Comparative functional analysis of the cow and mouse myostatin genes reveals novel regulatory elements in their upstream promoter regions

Myostatin is a paracrine/autocrine factor that inhibits muscle growth, and mutations that affect myostatin activity or expression produce dramatic increases in muscle mass in several species. However, at present it is less clear whether differences in myostatin expression or activity exist between species with differing body sizes. Here we demonstrate that mouse muscle expresses far greater levels of myostatin mRNA than cow. In addition, activity of a 1200 bp mouse myostatin promoter construct was significantly greater than that of a 1200 bp cow myostatin promoter construct in C₂C₁₂ myotubes. In contrast, activity of reporter constructs flanked by one or both untranslated regions (UTRs) was not significantly different between the two species. Sequence analysis identified a number of promoter regions which differed between larger species (cow, pig, goat, sheep, human) and smaller (mouse, rat), including a TATA-box sequence, a CACCC box, two AT-rich regions (AT1 and AT2), and a palindromic sequence (PAL). We therefore used mutagenesis to alter the mouse sequence for each of these elements to that of the cow. Mutagenesis of the TATA, CACC, and AT1 sequences of the mouse to those of the cow significantly decreased activity of the mouse myostatin promoter compared to the wild type mouse promoter, while mutation of the AT2 and PAL sequences tended to increase promoter activity. Finally, the cow myostatin promoter was less responsive to FoxO signaling than the mouse myostatin promoter. Together these data support the hypothesis that differences in promoter activity between mouse and cow may contribute to differences in expression of the myostatin gene between these species.     

Identification of candidate genes at quantitative trait loci on chicken chromosome Z using orthologous comparison of chicken, mouse, and human genomes

This study was undertaken to identify novel candidate genes at quantitative trait loci (QTL) on chicken chromosome Z (GGAZ) by comparing orthologous regions of chicken, human and mouse genomes. Primer sequences from marker flanking QTL positions (https://acedb.asg.wur.nl/) were obtained from www.iastate.edu/chickmap and blasted against the chicken genome (www.ensembl.org) using BLASTN. The best matches were those with the highest score, lowest E-values and highest percent identity. Orthologous regions in mice and humans, together with genes located on or around those loci were identified using the Ensembl website. Forty-six chicken genes, 91 mouse genes and 60 human genes associated with QTL on GGAZ were identified in the current study. Among the most promising candidate genes for egg production and egg shell quality are annexin A1 (ANXA1), osteoclast stimulating factor (OSF), thrombospondin-4 (THBS4), programmed cell death proteins (PDCD), follistatin (FST), growth hormone receptor (GHR), interferon (IFN) alpha and beta. The chicken IFN alpha and beta were located on GGAZ around position 13,000,000 bp on the draft chicken sequence map. The neuronal nicotinic acetylcholine receptor (nAChR) is located at a QTL region for abdominal fat (GGAZ 25483091 bp). Nicotine is an agonist at the nAChRs and has been shown to decrease lipolysis and triglyceride uptake, thereby reducing net storage in adipose tissue. Therefore, the nAchRs could be used as therapeutic targets for regulating feed intake and obesity. This study has identified 197 putative candidate genes in probable QTL regions of chicken chromosome Z.     

Localization of human X chromosomal mental retardation (MRX) genes in chicken and comparison with the chicken genome sequence data

In an ongoing study human X chromosomal mental retardation genes (MRX) were mapped in the chicken genome. Up to now the homologs of 13 genes were localized by FISH techniques. Four genes from HSAXp (TM4SF2, RSK2/RPS6KA3, NLGN4, ARX) map to GGA1q13-->q31, and seven genes from HSAXq (OPHN1, AGTR2, ARHGEF6, PAK3, FACL4/ACS4, FMR2, ATRX) to GGA4p. The gene-rich region of HSAXq28 proved to be much less conserved. GDI1 localized to GGA1pter and SLC6A8 to a mid-sized microchromosome. The order of the genes was determined from the newly available genome sequence data from chicken, which reveals exact colinearity between the genes in HSAXp and GGA1q13-->q31, but completely scrambled gene order between the genes with common synteny from HSAXq and GGA4p. This result supports the hypothesis that the human X chromosome is a real ancient autosomal linkage group.     

Cloning and sequence analysis reveal structural variation among related zein genes in maize

We have isolated a gene encoding one of the 19,000 dalton zein proteins from a maize genomic library constructed in Charon 4A. This gene occurs on a 7.7 kb Eco RI fragment, and based on Southern hybridization analysis, represents one of several homologous sequences present in the maize genome. The nucleotide sequence of the gene predicts a protein composed of 235 amino acids, including a signal peptide of 21 amino acids. There are no intervening sequences in the gene. By comparing the nucleotide sequence of this gene with that of a homologous cDNA clone, we have identified a basis for microheterogeneity within the gene family. The 5′ nucleotide sequences of the genomic and cDNA clones are identical, but they differ in the center of the protein, where repeated amino acid sequences occur. A nucleotide sequence encoding a conserved peptide of 20 amino acids is repeated nine times in the center of both of these clones.     

Cloning and sequence analysis of hydrogenase regulatory genes (hydHG) from Salmonella typhimurium.

The nucleotide sequence of the hydHG operon, comprised of chromosomal genes that regulate labile hydrogenase activity in Salmonella typhimurium, was compared with the reported hydHG sequence of Escherichia coli. Nucleotide sequence analysis of a 4.8 kb EcoRI fragment of Salmonella chromosomal DNA revealed that one of the open reading frames (ORF) encoded a protein of 441 amino acid residues. This large ORF was identified on a 2.7 kb Eco RI/HindIII fragment and coded for the complete hydG gene. The carboxy-terminus (626 bp) of the hydH gene also was present immediately upstream of hydG. Expression of the Salmonella hydG gene in a T7 promoter/polymerase system revealed the presence of a unique 45 kDa protein band. The incomplete hydH gene was not expressed. It is proposed that the labile hydrogenase activity in S. typhimurium may be regulated by the multiple component system.     

Cloning and chromosome mapping of human and chicken Iroquois (IRX) genes.

Three highly homologous homeobox genes (caupolican, araucan and mirror) have been identified in Drosophila. These genes belong to the novel Iroquois complex, which acts as a pre-pattern molecule in Drosophila neurogenesis. Recently several vertebrate Iroquois homologues (Irx) were isolated and found to be involved in pattern formation of various tissues. Here we report cytogenetic mapping of four human and five chicken Iroquois genes by FISH. Our findings revealed that vertebrate Irx genes are clustered at two different loci.     

Strategy for statistical-mapping of potential regulatory regions in the human genome

We have explored the feasibility of using crude nuclear extracts and band-shift experiments, to select and clone short human DNA fragments that contain binding sites for sequence-specific nuclear proteins. We show that this strategy is feasible. It provides a means to systematically identify the recognition sequences of proteins found in nuclear extracts prepared from various tissues, in order to compile a protein-binding-sequence catalogue. A complete catalogue would provide the data needed for searching the "raw" human DNA sequences for occurrence of protein-binding sites and thus, to construct a statistical map of potential regulatory regions in the human genome.