Cloning, sequencing, and analysis of inv8 chromosome breakpoints associated with recombinant 8 syndrome.

Rec8 syndrome (also known as "recombinant 8 syndrome" and "San Luis Valley syndrome") is a chromosomal disorder found in individuals of Hispanic descent with ancestry from the San Luis Valley of southern Colorado and northern New Mexico. Affected individuals typically have mental retardation, congenital heart defects, seizures, a characteristic facial appearance, and other manifestations. The recombinant chromosome is rec(8)dup(8q)inv(8)(p23.1q22.1), and is derived from a parental pericentric inversion, inv(8)(p23.1q22.1). Here we report on the cloning, sequencing, and characterization of the 8p23.1 and 8q22 breakpoints from the inversion 8 chromosome associated with Rec8 syndrome. Analysis of the breakpoint regions indicates that they are highly repetitive. Of 6 kb surrounding the 8p23.1 breakpoint, 75% consists of repetitive gene family members-including Alu, LINE, and LTR elements-and the inversion took place in a small single-copy region flanked by repetitive elements. Analysis of 3.7 kb surrounding the 8q22 breakpoint region reveals that it is 99% repetitive and contains multiple LTR elements, and that the 8q inversion site is within one of the LTR elements.

Human phosphoribosylformylglycineamide amidotransferase (FGARAT): regional mapping, complete coding sequence, isolation of a functional genomic clone, and DNA sequence analysis.

Purines play essential roles in many cellular functions, including DNA replication, transcription, intra- and extra-cellular signaling, energy metabolism, and as coenzymes for many biochemical reactions. The de-novo synthesis of purines requires 10 enzymatic steps for the production of inosine monophosphate (IMP). Defects in purine metabolism are associated with human diseases. Further, many anticancer agents function as inhibitors of the de-novo biosynthetic pathway. Genes or cDNAs for most of the enzymes comprising this pathway have been isolated from humans or other mammals. One notable exception is the phosphoribosylformylglycineamide amidotransferase (FGARAT) gene, which encodes the fourth step of this pathway. This gene has been cloned from numerous microorganisms and from Drosophila melanogaster and C. elegans. We report here the identification of a human cDNA containing the coding region of the FGARAT mRNA and the isolation of a P1 clone that contains an intact human FGARAT gene. The P1 clone corrects the purine auxotrophy and protein deficiency of Chinese hamster ovary (CHO) cell mutants (AdeB) deficient in both the activity and the protein for FGARAT. The P1 clone was used to regionally map the FGARAT gene to chromosome region 17p13, a location consistent with our prior assignment of this gene to chromosome 17. A comparison of the DNA sequence of the human FGARAT and FGARAT DNA sequence from 17 other organisms is reported. The isolation of this gene means that DNA clones for all the 10 steps of IMP synthesis have been isolated from humans or other mammals.

The human GARS-AIRS-GART gene encodes two proteins which are differentially expressed during human brain development and temporally overexpressed in cerebellum of individuals with Down syndrome.

Purines are critical for energy metabolism, cell signalling and cell reproduction. Nevertheless, little is known about the regulation of this essential biochemical pathway during mammalian development. In humans, the second, third and fifth steps of de novo purine biosynthesis are catalyzed by a trifunctional protein with glycinamide ribonucleotide synthetase (GARS), aminoimidazole ribonucleotide synthetase (AIRS) and glycinamide ribonucleotide formyltransferase (GART) enzymatic activities. The gene encoding this trifunctional protein is located on chromosome 21. The enzyme catalyzing the intervening fourth step of de novo purine biosynthesis, phosphoribosylformylglycineamide amidotransferase (FGARAT), is encoded by a separate gene on chromosome 17. To investigate the regulation of these proteins, we have generated monoclonal and/or polyclonal antibodies specific to each of these enzymatic domains. Using these antibodies on western blots of Chinese hamster ovary (CHO) cells transfected with the human GARS-AIRS-GART gene, we show that this gene encodes not only the trifunctional protein of 110 kDa, but also a monofunctional GARS protein of 50 kDa. This carboxy-truncated human GARS protein is produced by alternative splicing resulting in the use of a polyadenylation site in the intron between the terminal GARS and the first AIRS exons. The expression of both the GARS and GARS-AIRS-GART proteins are regulated during development of the human cerebellum, while the expression of FGARAT appears to be constitutive. All three proteins are expressed at high levels during normal prenatal cerebellum development while the GARS and GARS-AIRS-GART proteins become undetectable in this tissue shortly after birth. In contrast, the GARS and GARS-AIRS-GART proteins continue to be expressed during the postnatal development of the cerebellum in individuals with Down syndrome.

Localization of the L-glutamine synthetase gene to chromosome 1q23.

Glutamine synthetase (E.C. 6.3.1.2) is expressed throughout the body and plays an important role in controlling body pH and in removing ammonia from the circulation. The enzyme clears L-glutamate, the major neurotransmitter in the central nervous system, from neuronal synapses. The enzyme is a very sensitive marker of many disease and aging processes, especially those involving reactive oxygen species. This report describes the localization of the enzyme to chromosome 1 by PCR analysis of a human/rodent somatic cell hybrid panel. We also describe the localization of a recently described pseudogene to chromosome 9. Further localization of the glutamine synthetase gene locus to 1q23 was accomplished by fluorescence in situ hybridization. The glutamine synthetase gene was mapped to five CEPH megaYACs between the polymorphic PCR markers D1S117 and D1S466 by analysis of the Whitehead Institute's recently described chromosome 1 contig map.

Localization of the squalene synthase gene (FDFT1) to human chromosome 8p22-p23.1.

Recently, we reported the isolation of a cDNA encoding the human enzyme squalene synthase, the first step of sterol biosynthesis uniquely committed to synthesis of cholesterol (6). As such, it is likely that this enzyme occupies a critical regulatory position in the synthesis of cholesterol. As part of continuing studies of the role of this gene in cellular metabolism, we undertook the mapping of this gene on the human chromosomes. To localize the gene, we have first isolated a yeast artificial chromosome (YAC) containing the squalene synthase gene. We then used fluorescence in situ hybridization (FISH) with yeast DNA containing the YAC to localize the gene to chromosome 8. Assignment to human chromosome 8 was confirmed by polymerase chain reaction analysis of a somatic cell hybrid containing human chromosome 8. Use of a somatic cell hybrid regional mapping panel dividing chromosome 8 into several fragments localized the gene to 8p21-pter. Fractional length analysis of the FISH mapping placed the signal generated with this YAC at 8p22-p23.1.

Purification of, generation of monoclonal antibodies to, and mapping of phosphoribosyl N-formylglycinamide amidotransferase.

5'-Phosphoribosyl N-formylglycinamide (FGAR) amidotransferase (EC 6.3.5.3) catalyzes the fourth reaction in the de novo synthesis of purines, that is, the conversion of FGAR to 5'-phosphoribosyl N-formylglycinamidine (FGAM). This is the only step of the pathway for which a vertebrate gene has not been cloned. FGAR amidotransferase has been highly purified from Chinese hamster ovary (CHO) cells, and this preparation has been used to generate monoclonal antibodies in mice. Two of these antibodies, designated BD4 and DD2, have been shown to recognize a 150-kDa protein in CHO-K1 cells that is of very low abundance in Ade-B cells, a CHO line in which FGAR amidotransferase activity is undetectable. Furthermore, the protein recognized by these antibodies is 5-10-fold more abundant in Azr cells. The CHO Azr cell line was made resistant to azaserine, a potent inhibitor of FGAR amidotransferase, and displays a 5-10-fold increase in FGAR amidotransferase activity over the parental K1 line. FGAR amidotransferase activity and the 150-kDa protein recognized by both monoclonal antibodies were found to immunoprecipitate concomitantly using antibody BD4. Monoclonal antibody DD2 cross-reacted with a human protein of identical molecular mass. A number of Ade-B/human hybrid cells were generated by somatic cell fusion and subsequent 5-bromo-2-deoxyuridine segregation. Analysis of these lines, together with two independently generated human/mouse hybrid cell lines, by both cytogenetics and immunoblotting with antibody DD2 revealed that the human FGAR amidotransferase gene is located on chromosome 17p.

The human interleukin-1 receptor antagonist (IL1RN) gene is located in the chromosome 2q14 region.

The gene for human interleukin-1 receptor antagonist (IL1RN) has been assigned to chromosome 2 on the basis of Southern blot analysis of a series of human-Chinese hamster cell hybrids. Using a yeast artificial chromosome containing the IL1RN gene as a probe, the human IL1RN gene was localized to the long arm of chromosome 2 at band 2q14.2 by fluorescence in situ hybridization. This site is near the positions of genes for human IL-1 alpha, IL-1 beta, and types I and II IL-1 receptors, as reported by other laboratories.

A single base change at a splice acceptor site leads to a truncated CAD protein in Urd-A mutant Chinese hamster ovary cells.

We have previously reported the isolation and characterization of mutant Chinese hamster ovary (CHO-K1) cells of the Urd-A complementation group, which require uridine for growth, are deficient in the activities of the first three enzymes of de novo UMP biosynthesis, and produce markedly reduced amounts of a truncated form of the multifunctional protein CAD, which contains these three enzyme activities. We report here that a single base change of G to A at a highly conserved RNA splice acceptor site is responsible for the phenotype of this mutant. In addition to a small amount of apparently normal CAD mRNA, this mutation causes production of two alternative forms of CAD mRNA in the mutant, one that includes the intron just prior to the mutation and one that excludes the exon just after the mutation. The affected splice site is located at the intron-exon boundary just preceding the exon that encodes the beginning of the aspartate transcarbamylase (ATCase) domain of the CAD protein. Both intron inclusion and exon exclusion during RNA processing introduce a translation stop codon upstream of the region encoding this domain, resulting in the production of the truncated CAD protein seen in the Urd-A mutant. This mutation also results in markedly decreased levels of CAD mRNA and protein in the mutant.

Expression of a human cDNA encoding a protein containing GAR synthetase, AIR synthetase, and GAR transformylase corrects the defects in mutant Chinese hamster ovary cells lacking these activities.

The isolation of a human cDNA encoding the multifunctional protein containing GAR synthetase, AIR synthetase, and GAR transformylase by functional complementation of purine auxotrophy in yeast has been reported. Chinese hamster ovary (CHO) cell mutant purine auxotrophs deficient in GAR synthetase (Ade-C) or AIR synthetase plus GAR transformylase (Ade-G) activities were transfected with this human GART cDNA subcloned into a mammalian expression vector. This restored 49-140% of the activities of GAR synthetase, AIR synthetase, and GAR transformylase in transfected cells when compared to wild-type CHO K1 parental cells. Study of one stably expressing transfectant, AdeC2, revealed that the human GART cDNA was incorporated into the CHO genome. The enzyme activities appear to be associated with an expressed protein of 110 kDa, very similar to that of purified human GART trifunctional enzyme. The Ade-C mutant shows reduced amounts of GART mRNA compared to CHO K1 and a protein of apparently reduced size, results consistent with the purine requirement and enzyme deficiency observed in the mutant. These experiments provide definitive evidence that the human GART cDNA encodes and can direct the production of active human GART trifunctional protein in mammalian cells. They also provide important evidence that the Ade-C and Ade-G mutants of CHO cells are defective in this gene.

Isolation of a human cDNA encoding amidophosphoribosyltransferase and functional complementation of a CHO Ade-A mutant deficient in this activity.

We report here the isolation of a human cDNA encoding the first step in de novo purine biosynthesis, amidophosphoribosyltransferase (PRAT). The human PRAT cDNA was isolated by complementation of a Saccharomyces cerevisiae ade4 mutant deficient in PRAT enzymatic activity. The identity of the isolated cDNA, designated pAdeA-3, was confirmed by several independent methods. Genomic DNA sequences homologous to pAdeA-3 show coordinate segregation with the hypoxanthine nutritional requirement in Chinese hamster ovary (CHO) cell Ade-A-human hybrids, segregants of these hybrids, and irradiation reduction hybrids. The PRAT cDNA after insertion into a mammalian expression vector was capable of correcting the PRAT cDNA after insertion into a mammalian expression vector was capable of correcting the PRAT enzyme deficiency in CHO Ade-A mutants. This correction was monitored by both cell-free PRAT assays and in vivo phosphoribosylformylglycinamide (FGAR) accumulation studies. FGAR accumulation is a classic method for assessment of the early steps of purine nucleotide biosynthesis. Two of the isolated transformants, designated PRAT-1 and PRAT-2, exhibited 22% and 53%, respectively, of wild-type CHO K1 PRAT enzymatic activity using a cell-free enzyme assay. These same two transformants plus an additional transformant, designated PRAT-13, showed FGAR accumulations of 150%, 260%, and 140%, respectively, compared to the levels of accumulation seen in CHO K1. Transformants PRAT-1 and PRAT-2 both contained a mRNA species recognized by the PRAT cDNA of identical size to a mRNA species in human fibroblasts homologous to the PRAT cDNA. This observation, along with the functionality of the cDNA in both yeast and CHO cells deficient in PRAT activity, suggests the isolated cDNA is full length.

Drosophila purine auxotrophy: new alleles of adenosine 2 exhibiting a complex visible phenotype.

New mutant alleles of the adenosine2 locus (ade2; 2-17.7) have been isolated using the eye-color phenotype exhibited by the prototype auxotrophic allele ade2 as the screening criterion. The new mutants form a single complementation group, suggesting that they all exhibit purine auxotrophy and defective formylglycineamide ribotide amidotransferase enzyme, like ade2. Tests carried out on particular new alleles confirm these suggestions. The new mutants all exhibit more extreme physical defects than the prototype. They have wing abnormalities like mutants defective in pyrmidine biosynthesis and reduced bristles like those defective in protein synthesis; thus they exhibit the combined visible phenotype of rudimentary wings, rosy eyes, and bobbed bristles. Cytogenetic analysis places the locus in the interband proximal to 26B1-2.

Two Drosophila melanogaster mutations block successive steps of de novo purine synthesis.

Drosophila melanogaster purine auxotrophs ade2(1) and ade3(1) have been characterized biochemically. The ade2(1) strain is deficient in the fourth step of the de novo purine synthetic pathway catalyzed by phosphoribosylglycinamidine synthase (phosphoribosylformylglycinamide amidotransferase). The ade3(1) strain is deficient in the previous step catalyzed by phosphoribosylglycinamide formyltransferase (GART). The mutation responsible for the slightly leaky ade3(1) phenotype was characterized further. First, the mutant GART polypeptide was found to be of normal size and present at normal levels. Second, the GART-encoding region of the mutant was cloned, inserted into a yeast-Escherichia coli shuttle vector, and used to transform mutant yeast. Transformants showed very slight in vivo activity when compared to wild type, verifying that the mutation is in the GART coding sequence. Lastly, the region of the gene encoding GART activity from mutant and inbred parental strain flies was completely sequenced. A single base transition was found, leading to the substitution of a serine for a highly conserved glycine. These two mutations provide examples of blocks in the de novo purine synthetic pathway in a whole animal.

Multiple purine pathway enzyme activities are encoded at a single genetic locus in Drosophila.

The Drosophila melanogaster Gart locus, known from previous work to encode the enzyme activity phosphoribosylglycinamide formyltransferase (GART), specifies two alternatively processed mRNAs and two proteins. We introduced the entire Gart locus into a Drosophila tissue culture cell line in which the locus is active. The resulting cell clones contained numerous copies of the locus and overproduced both mRNAs and both expected proteins, thus markedly facilitating analysis of these molecules. We assayed extracts of the clones for the activities of 10 enzymes important for de novo purine synthesis and found that, in addition to GART, two other purine pathway activities, phosphoribosylamine-glycine ligase (phosphoribosylglycinamide synthetase, GARS) and phosphoribosylformylglycinamidine cyclo-ligase (phosphoribosylaminoimidazole synthetase, AIRS), are similarly overproduced. All three activities are present together on the larger overproduced protein. A smaller protein appears to possess only GARS activity. Therefore, alternative mRNA processing can allow cells to produce enzyme activities in forms that are either linked or unlinked to other activities.

Translocation of c-myc in the hereditary renal cell carcinoma associated with a t(3;8)(p14.2;q24.13) chromosomal translocation.

A translocation between chromosomes 3 and 8, t(3;8)(p14.2;q24.13), has been reported in a family with hereditary renal cell carcinoma. Using somatic cell hybrids, we have isolated, separately, both derivative chromosomes. We find that the c-myc oncogene (8q24.1) has been translocated to the derivative 3 [der(3)]. We have not detected a rearrangement within an approximately equal to 21-kilobase region around the c-myc gene using restriction enzyme digestion and Southern blot hybridization analysis. The translocated c-myc gene should provide a probe to the chromosome 3p14 region, which appears to be important not only in renal cell carcinoma but also in small cell carcinoma of the lung. These hybrids have also been useful for the regional mapping of the Chinese hamster ovary cell Gly-B defect to 8q22.1----q24.13 and support the regional assignment of acylase I to 3p21.