DLAD, a novel mammalian divalent cation-independent endonuclease with homology to DNase II.

In this report, we describe the molecular cloning and characterization of DLAD, a novel mammalian deoxy-ribonuclease homologous to DNase II. The full length cDNA for mouse DLAD has been cloned by polymerase chain reaction. The cDNA contains a 1065 bp open reading frame (ORF) encoding a 354 amino acid protein with a calculated molecular mass of 40 767. The predicted protein for DLAD shares 34.4% identity with DNase II. DLAD is also homologous to three predicted proteins, C07B5.5, F09G8.2 and K04H4.6, from the nematode Caenorhabditis elegans. Furthermore, the third ORF of the fowlpox virus genome is found to encode a DLAD homologue showing 37. 1% identity at the amino acid level. Northern blot analysis reveals that expression of the DLAD mRNA is highly restricted to the liver. DLAD mainly exists as a cytoplasmic protein with divalent cation-independent endonuclease activity and cleaves DNA to produce 3'-phosphoryl/5'-hydroxyl ends. It is active under a wide range of pH with maximum activity at pH 5.2. Among known DNase inhibitors tested, aurintricarboxylic acid and Zn(2+)are found to be effective inhibitors of the DLAD activity.     

HSF4 regulates DLAD expression and promotes lens de-nucleation

HSF4 mutations lead to both congenital and age-related cataract. The purpose of this study was to explore the mechanism of cataract formation caused by HSF4 mutations. The degradation of nuclear DNA is essential for the lens fiber differentiation. DNase 2β (DLAD) is highly expressed in lens cells, and mice with deficiencies in the DLAD gene develop nuclear cataracts. In this study, we found that HSF4 promoted the expression and DNase activity of DLAD by directly binding to the DLAD promoter. In contrast, HSF4 cataract causative mutations failed to bind to the DLAD promoter, abrogating the expression and DNase activity of DLAD. These results were confirmed by HSF4 knockdown in zebrafish, which led to incomplete de-nucleation of the lens and decreased expression and activity of DLAD. Together, our results suggest that HSF4 exerts its function on lens differentiation via positive regulation of DLAD expression and activity, thus facilitating de-nucleation of lens fiber cells. Our demonstration that HSF4 cataract causative mutations abrogate the induction of DLAD expression reveals a novel molecular mechanism regarding how HSF4 mutations cause cataractogenesis.     

Structure and sequence of the human homeobox gene HOX7.

A cosmid containing the human sequence HOX7, homologous to the murine Hox-7 gene, was isolated from a genomic library, and the positions of the coding sequences were determined by hybridization. DNA sequence analysis demonstrated two exons that code for a homeodomain-containing protein of 297 amino acids. The open reading frame is interrupted by a single intron of approximately 1.6 kb, the splice donor and acceptor sites of which conform to known consensus sequences. The human HOX7 coding sequence has a very high degree of identity with the murine Hox-7 cDNA. Within the homeobox, the two sequences share 94% identity at the DNA level, all substitutions being silent. This high level of sequence similarity is not confined to the homeodomain; overall the human and murine HOX7 gene products show 80% identity at the amino acid level. Both the 5' and 3' untranslated regions also show significant similarity to the murine gene, with 79 and 70% sequence identity, respectively. The sequence upstream of the coding sequence of exon 1 contains a GC-rich putative promoter region. There is no TATA box, but a CCAAT and numerous GC boxes are present. The region encompassing the promoter region, exon 1, and the 5' region of exon 2 have a higher than expected frequency of CpG dinucleotides; numerous sites for rare-cutter restriction enzymes are present, a characteristic of HTF islands.     

Cloning and characterization of an actin-resistant DNase I-like endonuclease secreted by macrophages

We have cloned human and murine DNase I-like cDNAs, termed LS-DNase, which are expressed at high levels in liver and spleen tissues. LS-DNase expression is highly specific to macrophage populations within these and other tissues. Mature LS-DNase from both species is a secreted, non-glycosylated protein containing 285 residues, with a calculated molecular mass of 33 kDa and a basic isolelectric point. Human and murine LS-DNase are highly conserved and share 83% identity. Sequence analysis reveals that LS-DNase shares 46% amino acid sequence identity with DNase I. However, several residues identified as important for interaction of human DNase I with actin are not conserved in both human and murine LS-DNase. Consistent with this observation, recombinant human LS-DNase possesses a DNA hydrolytic activity which, unlike DNase I, is not inhibited by G-actin. The existence of a family of DNase I-like molecules that have tissue-specific expression patterns and the possible role of a macrophage specific DNase are discussed.     

The genomic sequence of the murine major vault protein and its promoter

Vaults are ribonucleoproteins of unknown function, consisting of three different proteins and multiple copies of small untranslated RNA molecules. One of the protein subunits has been identified as TEP1, a protein that is also associated with the telomerase complex. Another protein appears to contain a functional PARP domain and is hence called VPARP. The third protein, major vault protein (MVP), is believed to make up 70% of the total mass of the vault complex and to be responsible for the typical barrel-shaped structure of vaults. We have isolated the murine MVP cDNA and compared the amino acid sequence with MVP from other species. Over 90% of sequence identity was found between mouse, human and rat, and a considerable degree of identity between mouse and MVPs from lower eukaryotes. We also found that the genomic structure of the murine MVP gene closely resembles the organization of the human MVP gene, both consisting of 15 exons of which most have exactly the same size. Finally we have isolated a genomic region upstream (and partially overlapping) the first untranslated exon, that displayed promoter activity in a luciferase reporter assay. Furthermore, we showed that the sequences from the first exon together with the 5'-end of the first intron enhance the promoter activity, implying the presence of essential promoter elements in this region. Alignment of the murine promoter region with the homologous sequences of the human gene revealed an identity of 58%. The apparent presence of conserved promoter elements suggests a similar regulation of human and murine MVP expression.     

The cloning, genomic structure, localization, and expression of human deoxyribonuclease IIbeta

Acidic endonuclease activity is present in all cells in the body and much of this can be attributed to the previously cloned and ubiquitously expressed deoxyribonuclease II (DNase II). Database analysis revealed the existence of expressed sequence tags and genomic segments coding for a protein with considerable homology to DNase II. This report describes the cloning of this cDNA, which we term deoxyribonuclease IIbeta (DNase IIbeta) and comparison of its expression to that of the originally cloned DNase II (now termed DNase IIalpha). The cDNA encodes a 357 amino acid protein. This protein exhibits extensive homology to DNase IIalpha including an amino-terminal signal peptide and a conserved active site, and has many of the regions of identity that are conserved in homologs in other mammals as well as C. elegans and Drosophila. The gene encoding DNase IIbeta has identical splice sites to DNase IIalpha. Human DNase IIbeta is highly expressed in the salivary gland, and at low levels in trachea, lung, prostate, lymph node, and testis, whereas DNase IIalpha is ubiquitously expressed in all tissues. The expression pattern of human DNase IIbeta suggests that it may function primarily as a secreted enzyme. Human saliva was found to contain DNase IIalpha, but after immunodepletion, considerable acid-active endonuclease remained which we presume is DNase IIbeta. We have localized the gene for human DNase IIbeta to chromosome 1p22.3 adjacent (and in opposing orientation) to the human uricase pseudogene. Interestingly, murine DNase IIbeta is highly expressed in the liver. Uricase is also highly expressed in mouse but not human liver and this may explain the difference in expression patterns between human and mouse DNase IIbeta.     

Gene Structure, Chromosomal Location, and Expression Pattern of Maleylacetoacetate Isomerase

The gene for maleylacetoacetate isomerase (MAAI) (EC 5.2.1.2) was the last gene in the mammalian phenylalanine/tyrosine catabolic pathway to be cloned. We have isolated the human and murine genes and determined their genomic structure. The human gene spans a genomic region of approximately 10 kb, has 9 exons ranging from 50 to 528 bp in size, and was mapped to 14q24.3-14q31.1 using fluorescence in situ hybridization. The complete catabolic pathway of phenylalanine/tyrosine is normally restricted to liver and kidney, but the maleylacetoacetate isomerase gene is expressed ubiquitously. This suggests a possible second role for the MAAI protein different from phenylalanine/tyrosine catabolism. We have searched for mutations in the maleylacetoacetate isomerase gene in four cases of unexplained severe liver failure in infancy with clinical similarities to hereditary tyrosinemia type I (pseudotyrosinemia). Several amino acid changes were identified, but all were found to retain MAAI activity and thus represent protein polymorphisms. We conclude that MAAI deficiency is not a common cause of the pseudotyrosinemic phenotype.     

Genomic structure and comparison of mouse tissue-specific alkaline phosphatase genes

A full-length human placental alkaline phosphatase (AP) cDNA was used to identify and clone related genes from mouse genomic libraries. We report the cloning, sequence, and structural comparison of the mouse embryonic and intestinal AP genes and a putative AP pseudogene. All three mouse genes are composed of 11 exons interrupted by 10 small introns (70-261 bp) with an organization analogous to that of the three human tissue-specific AP genes. Introns interrupt the coding sequences at identical positions in all three mouse and human tissue-specific AP genes. The deduced amino acid sequence of the isozymes predicts proproteins of 529, 559, and 466 amino acids for embryonic AP, intestinal AP, and pseudo-AP, respectively. A repetitive sequence inserted in exon XI of the mouse intestinal AP gene codes for a unique stretch of 41 amino acids, 20 of which are threonines. This insertion has disrupted a region recognized as being responsible for phosphatidylinositol anchorage of human placental AP to the cytoplasmic membrane. Phylogenetic analysis indicates that the three mouse AP isozymes form a distinct group separate from the human tissue-specific AP isozymes, suggesting the taxon-specific evolution of the AP genes as opposed to independent evolution of AP genes expressed in specific tissues.     

Intron loss in the SART1 genes of Fugu rubripes and Tetraodon nigroviridis.

The human SART1 gene was initially identified in a screen for proteins recognised by IgE, which may be implicated in atopic disease. We have examined the genomic structure and cDNA sequence of the SART1 gene in the compact genomes of the pufferfish Fugu rubripes and Tetraodon nigroviridis. The entire coding regions of both the Fugu and Tetraodon SART1 genes are contained within single exons. The Fugu gene contains only one intron located in the 5' untranslated region. Southern blot hybridisation of Fugu genomic DNA confirmed the SART1 gene to be single copy. Partial genomic structures were also determined for the human, mouse, Drosophila and C. elegans SART1 homologues. The human and mouse genes both contain many introns in the coding region, the human gene possessing at least 20 exons. The Drosophila and C. elegans homologues contain 6 and 12 exons, respectively. This is only the second time such a difference in the organization of homologous Fugu and human genes has been reported. The Fugu and Tetraodon SART1 genes encode putative proteins of 772 and 774 aa, respectively, each having 65% amino acid identity to human SART1. Leucine zipper and basic motifs are conserved in the predicted Fugu and Tetraodon proteins.