The human gamma-glutamyltransferase gene family

Assays for gamma-glutamyl transferase (GGT1, EC 2.3.2.2) activity in blood are widely used in a clinical setting to measure tissue damage. The well-characterized GGT1 is an extracellular enzyme that is anchored to the plasma membrane of cells. There, it hydrolyzes and transfers γ-glutamyl moieties from glutathione and other γ-glutamyl compounds to acceptors. As such, it has a critical function in the metabolism of glutathione and in the conversion of the leukotriene LTC4 to LTD4. GGT deficiency in man is rare and for the few patients reported to date, mutations in GGT1 have not been described. These patients do secrete glutathione in urine and fail to metabolize LTC4. Earlier pre-genome investigations had indicated that besides GGT1, the human genome contains additional related genes or sequences. These sequences were given multiple different names, leading to inconsistencies and confusion. Here we systematically evaluated all human sequences related to GGT1 using genomic and cDNA database searches and identified thirteen genes belonging to the extended GGT family, of which at least six appear to be active. In collaboration with the HUGO Gene Nomenclature Committee (HGNC) we have designated possible active genes with nucleotide or amino acid sequence similarity to GGT1, as GGT5 (formerly GGL, GGTLA1/GGT-rel), GGT6 (formerly rat ggt6 homologue) and GGT7 (formerly GGTL3, GGT4). Two loci have the potential to encode only the light chain portion of GGT and have now been designated GGTLC1 (formerly GGTL6, GGTLA4) and GGTLC2. Of the five full-length genes, three lack of significant nucleotide sequence homology but have significant (GGT5, GGT7) or very limited (GGT6) amino acid similarity to GGT1 and belong to separate families. GGT6 and GGT7 have not yet been described, raising the possibility that leukotriene synthesis, glutathione metabolism or γ-glutamyl transfer is regulated by their, as of yet uncharacterized, enzymatic activities. In view of the widespread clinical use of assays that measure γ-glutamyl transfer activity, this would appear to be of significant interest. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The human HOX gene family.

We report the identification of 10 new human homeobox sequences. Altogether, we have isolated and sequenced 30 human homeoboxes clustered in 4 chromosomal regions called HOX loci. HOX1 includes 8 homeoboxes in 90 kb of DNA on chromosome 7. HOX2 includes 9 homeoboxes in 180 kb on chromosome 17. HOX3 contains at least 7 homeoboxes in 160 kb on chromosome 12. Finally, HOX4 includes 6 homeoboxes in 70 kb on chromosome 2. Homeodomains obtained from the conceptual translation of the isolated homeoboxes can be attributed to 13 homology groups on the basis of their primary peptide sequence. Moreover, it is possible to align the 4 HOX loci so that corresponding homeodomains in all loci share the maximal sequence identity. The complex of these observations supports and extends an evolutionary hypothesis concerning the origin of mammalian and fly homeobox gene complexes. We also determined the coding region present in 3 HOX2 cDNA clones corresponding to HOX2G, HOX2H and HOX2I.     

The human protein disulfide isomerase gene family

Enzyme-mediated disulfide bond formation is a highly conserved process affecting over one-third of all eukaryotic proteins. The enzymes primarily responsible for facilitating thiol-disulfide exchange are members of an expanding family of proteins known as protein disulfide isomerases (PDIs). These proteins are part of a larger superfamily of proteins known as the thioredoxin protein family (TRX). As members of the PDI family of proteins, all proteins contain a TRX-like structural domain and are predominantly expressed in the endoplasmic reticulum. Subcellular localization and the presence of a TRX domain, however, comprise the short list of distinguishing features required for gene family classification. To date, the PDI gene family contains 21 members, varying in domain composition, molecular weight, tissue expression, and cellular processing. Given their vital role in protein-folding, loss of PDI activity has been associated with the pathogenesis of numerous disease states, most commonly related to the unfolded protein response (UPR). Over the past decade, UPR has become a very attractive therapeutic target for multiple pathologies including Alzheimer disease, Parkinson disease, alcoholic and non-alcoholic liver disease, and type-2 diabetes. Understanding the mechanisms of protein-folding, specifically thiol-disulfide exchange, may lead to development of a novel class of therapeutics that would help alleviate a wide range of diseases by targeting the UPR.     

The shape of human gene family phylogenies

Background  

The shape of phylogenetic trees has been used to make inferences about the evolutionary process by comparing the shapes of actual phylogenies with those expected under simple models of the speciation process. Previous studies have focused on speciation events, but gene duplication is another lineage splitting event, analogous to speciation, and gene loss or deletion is analogous to extinction. Measures of the shape of gene family phylogenies can thus be used to investigate the processes of gene duplication and loss. We make the first systematic attempt to use tree shape to study gene duplication using human gene phylogenies.     

High sequence similarity within ras exons 1 and 2 in different mammalian species and phylogenetic divergence of the ras gene family

We have determined the canine and feline N-, K-, and H-ras gene sequences from position +23 to +270 covering exons I and II which contain the mutational hot spot codons 12, 13, and 61. The results were used to assess the degree of similarity between ras gene DNA regions containing the critical domains affected in neoplastic disorders in different mammalian species. The comparative analyses performed included human, canine, feline, murine, rattine, and, whenever possible, bovine, leporine (rabbit), porcelline (guinea pig), and mesocricetine (hamster) ras gene sequences within the region of interest. Comparison of feline and canine nucleotide sequences with the corresponding regions in human DNA revealed a sequence similarity greater than 85% to the human sequence. Contemporaneous analysis of previously published ras DNA sequences from other mammalian species showed a similar degree of homology to human DNA. Most nucleotide differences observed represented synonymous changes without effect on the amino acid sequence of the respective proteins. For assessment of the phylogenetic evolution of ras gene family, a maximum parsimony dendrogram based on multiple sequence alignment of the common region of exons I and II in the N-, K-, and H-ras genes was constructed. Interestingly, a higher substitution rate among the H-ras genes became apparent, indicating accelerated sequence evolution within this particular clade. The most parsimonious tree clearly shows that the duplications giving rise to the three ras genes must have occurred before the mammalian radiation. Received: 23 July 1997 / Accepted: 30 October 1997     

Epigenetic inactivation of the Ras-association domain family 1 (RASSF1A) gene and its function in human carcinogenesis

The Ras GTPases are a superfamily of molecular switches that regulate cellular proliferation and apoptosis in response to extra-cellular signals. The regulation of these pathways depends on the interaction of the GTPases with specific effectors. Recently, we have cloned and characterized a novel gene encoding a putative Ras effector: the Ras-association domain family 1 (RASSF1) gene. The RASSF1 gene is located in the chromosomal segment of 3p21.3. The high allelic loss in a variety of cancers suggested a crucial role of this region in tumorigenesis. At least two forms of RASSF1 are present in normal human cells. The RASSF1A isoform is highly epigenetically inactivated in lung, breast, ovarian, kidney, prostate, thyroid and several other carcinomas. Re-expression of RASSF1A reduced the growth of human cancer cells supporting a role for RASSF1 as a tumor suppressor gene. RASSF1A inactivation and K-ras activation are mutually exclusive events in the development of certain carcinomas. This observation could further pinpoint the function of RASSF1A as a negative effector of Ras in a pro-apoptotic signaling pathway. In malignant mesothelioma and gastric cancer RASSF1A methylation is associated with virus infection of SV40 and EBV, respectively, and suggests a causal relationship between viral infection and progressive RASSF1A methylation in carcinogenesis. Furthermore, a significant correlation between RASSF1A methylation and impaired lung cancer patient survival was reported, and RASSF1A silencing was correlated with several parameters of poor prognosis and advanced tumor stage (e.g. poor differentiation, aggressiveness, and invasion). Thus, RASSF1A methylation could serve as a useful marker for the prognosis of cancer patients and could become important in early detection of cancer.     

Multistage carcinogenesis induced by ras and myc oncogenes in a reconstituted organ

ras and myc oncogenes were able to induce distinct phenotypic alterations, resembling different types of premalignant lesions, when introduced into approximately 0.1% of the cells used to reconstitute the mouse prostate gland. While ras induced dysplasia in combination with angiogenesis, myc induced a hyperplasia of the otherwise normally developed organ. ras and myc together induced primarily carcinomas. However, tumor progression was also associated with additional genetic alterations involving gene amplification. Our data indicate that specific types of benign premalignant lesions may reflect the activation of different single oncogenes, and that the consecutive activation of multiple oncogenes could be a causal event in the step-like progression of tumorigenesis.     

The structure and evolution of the human salivary proline-rich protein gene family

We present the nucleotide sequences of four members of the six-member human salivary prolinerich protein (PRP) gene family. The four genes are PRB1 and PRB2, which encode basic PRPs, and PRB3 and PRB4, which encode glycosylated PRPs. Each PRB gene is approximately 4.0 kb in length and contains four exons, the third of which is entirely composed of 63-bp tandem repeats and encodes the proline-rich portion of the protein products. Exon 3 contains different numbers of tandem repeats in the different PRB genes. Variation in the numbers of these repeats is also responsible for length variations in different alleles of the PRB genes. We have determined a probable evolutionary history of the human PRP gene family by comparing the nucleotide sequences of the six PRP genes. The present-day six PRP loci probably evolved from a single ancestral gene by four sequential gene duplications, leading to six genes that fall into three subsets, each consisting of two genes. During this evolutionary process, multiple rearrangements and gene conversion occurred mainly in the region from the 3 end of IVS2 and the 3 end of exon 3.     

ras gene Amplification and malignant transformation.

Morphologic transformation of NIH 3T3 mouse cells occurs upon transfection of these cells with large amounts (greater than or equal to 10 micrograms) of recombinant DNA molecules carrying the normal human H-ras-1 proto-oncogene. We provide experimental evidence indicating that transformation of these NIH 3T3 cells results from the combined effect of multiple copies of the H-ras-1 proto-oncogene rather than from spontaneous mutation of one of the transfected H-ras-1 clones (E. Santos, E.P. Reddy, S. Pulciani, R.J. Feldman, and M. Barbacid, Proc. Natl. Acad. Sci. USA 80:4679-4683, 1983). Levels of H-ras-1 RNA and p21 expression are highly elevated in the NIH 3T3 transformants, and in those cases examined, these levels correlate with the malignant properties of these cells. We have also investigated the presence of amplified ras genes in a variety of human carcinomas. In 75 tumor biopsies, we found amplification of the human K-ras-2 locus in one carcinoma of the lung. These results indicate that ras gene amplification is an alternative pathway by which ras genes may participate in the development of human neoplasia.     

The lipase gene family

Development of the lipase gene family spans the change in science that witnessed the birth of contemporary techniques of molecular biology. Amino acid sequencing of enzymes gave way to cDNA cloning and gene organization, augmented by in vitro expression systems and crystallization. This review traces the origins and highlights the functional significance of the lipase gene family, overlaid on the background of this technical revolution. The gene family initially consisted of three mammalian lipases [pancreatic lipase (PL), lipoprotein lipase, and hepatic lipase] based on amino acid sequence similarity and gene organization. Family size increased when several proteins were subsequently added based on amino acid homology, including PL-related proteins 1 and 2, phosphatidylserine phospholipase A1, and endothelial lipase. The physiological function of each of the members is discussed as well as the region responsible for lipase properties such as enzymatic activity, substrate binding, heparin binding, and cofactor interaction. Crystallization of several lipase gene family members established that the family belongs to a superfamily of enzymes, which includes esterases and thioesterases. This superfamily is related by tertiary structure, rather than amino acid sequence, and represents one of the most populous families found in nature.     

The paraoxonase gene family and atherosclerosis

Epidemiologic, genetic, and biochemical studies support an antiatherogenic role for paraoxonase (PON) 1. While the precise mechanism by which PON1 protects against the development of atherosclerosis is unclear, in vitro studies and the results from PON1 knockout and transgenic mice suggest that this protective effect may be attributed to PON1's ability to attenuate the oxidative modification of lipoprotein particles. The two other members of the PON gene family, namely, PON2 and PON3, have also been reported to possess antioxidant properties and may exhibit antiatherogenic capacities as well. Previous studies have demonstrated that PON1 expression is downregulated by oxidative stress. In contrast, more recent studies have shown that PON2 expression is upregulated in response to oxidative stress-inducing agents, while PON3 expression remains unchanged. While the physiological function of these proteins is unknown, studies currently underway using PON2 and PON3 knockout and transgenic mice should enable us to tease out the apparently redundant functions of these three proteins and yield clues as to their physiological function as well as their role in atherogenesis.     

A transforming ras gene can provide an essential function ordinarily supplied by an endogenous ras gene.

Microinjection of monoclonal antibody Y13-259, which reacts with all known mammalian and yeast ras-encoded proteins, has previously been shown to prevent NIH 3T3 cells from entering the S phase (L. S. Mulcahy, M. R. Smith, and D. W. Stacey, Nature [London] 313:241-243, 1985). We have now found several transformation-competent mutant v-rasH genes whose protein products in transformed NIH 3T3 cells are not immunoprecipitated by this monoclonal antibody. These mutant proteins are, however, precipitated by a different anti-ras antibody. Each of these mutants lacks Met-72 of v-rasH. In contrast to the result for cells transformed by wild-type v-rasH, Y13-259 microinjection of NIH 3T3 cells transformed by these mutant ras genes did not prevent the cells from entering the S phase. These results imply that a transformation-competent ras gene can supply a normal essential function for NIH 3T3 cells. When the proteins encoded by the mutant ras genes were overproduced in Escherichia coli, several mutant proteins that lacked Met-72 failed to bind Y13-259 in a Western blot. However, a ras protein from a mutant lacking amino antibody, but a ras protein from a mutant lacking amino acids 72 to 84 did not. These results suggest that Y13-259 may bind to a higher ordered structure that has been restored in the mutant lacking amino acids 72 to 82.