The UPS locus encoding uroporphyrinogen I synthase is located on human chromosome 11

The expression of the UPS locus encoding uroporphyrinogen I synthase has been investigated in human/mouse somatic cell hybrids. Human and mouse uroporphyrinogen I synthase can be readily distinguished by their isoelectric points. In hybrid cells, both human and mouse isozymes are detected. The multiple human uroporphyrinogen I synthase isozymes segregate as a single unit, as expected if they are the products of a single locus. The absence of new heteropolymers in hybrid cells supports the biochemical evidence that the active enzyme is a monomer. The presence of human uroporphyrinogen I synthase in hybrid clones was correlated with the presence of human chromosome 11, or its enzymatic marker, without exception in 44 independent hybrid lines. All other chromosomes could be eliminated as possible locations for this locus, due to their independent segregation. This report represents the first gene assignment for an enzyme in the heme biosynthesis pathway.     

Assignment of the gene for human arylsulfatase B, ARSB, to chromosome region 5p11----5qter

The structural gene coding for human arylsulfatase B, ARSB, is assigned to 5p11----5qter by analysis of somatic cell hybrids isolated from two separate fusions of human fibroblasts carrying a translocation involving chromosome 5 with the Chinese hamster cell line a3.     

Assignment of human pepsinogen A locus to the q12-pter region of chromosome 11

Summary A 0.9 kb cDNA fragment, corresponding to a large part of Rhesus monkey pepsinogen A mRNA, was used as probe for the chromosomal localization of the human pepsinogen A gene(s) using human-rodent somatic cell hybrids. Southern blot analysis of 14 human-Chinese hamster and three human-mouse cell hybrids, strongly indicates that the human PGA locus is on chromosome 11. The human-mouse hybrids, containing a translocation involving chromosome 11, allow sublocalization to the region q12-pter.     

Assignment of uroporphyrinogen decarboxylase (UROD) to the pter----p21 region of human chromosome 1

The assignment of the human gene for uroporphyrinogen decarboxylase (UROD) to chromosome 1 is confirmed and further localized to the pter----p21 region through the use of human-mouse somatic cell hybrids. Human and mouse UROD were separated by electrophoresis and identified with antibodies to the human enzyme after electrophoretic transfer to nitrocellulose membranes.     

Assignment of the alpha B-crystallin gene to human chromosome 11

Using a human alpha B-crystallin genomic probe and human-mouse somatic cell hybrids, the human alpha B-gene was assigned to chromosome 11 and further corroborated by in situ hybridization to normal metaphase chromosomes. This assignment confirmed and regionally mapped the locus to q22.3-23.1.     

Assignment of the human parathyroid hormone gene to chromosome 11

Summary A panel of human-mouse and human-Chinese hamster cell hybrid DNA's was screened for hybridisation with a fragment of the human parathyroid hormone chromosomal gene. A 7-kilobasepair Msp I restriction fragment homologous to this probe was found to segregate with the human chromosome 11.     

Assignment of the pepsinogen gene complex (PGA) to human chromosome region 11q13 by in situ hybridization

The genes coding for human pepsinogen (PGA3, PGA4, and PGA5) were assigned to chromosome region 11q13 by in situ hybridization. Previously we localized the PGA gene complex to a centromeric region of chromosome 11 (p11----q13) by Southern blot analysis of mouse-human somatic cell hybrids. Our in situ hybridization results confirm this assignment and further localize the genes to a smaller region on the long arm.     

Assignment of the human dihydrofolate reductase gene to the q11----q22 region of chromosome 5.

Cells from a dihydrofolate reductase-deficient Chinese hamster ovary cell line were hybridized to human fetal skin fibroblast cells. Nineteen dihydrofolate reductase-positive hybrid clones were isolated and characterized. Cytogenetic and biochemical analyses of these clones have shown that the human dihydrofolate reductase (DHFR) gene is located on chromosome 5. Three of these hybrid cell lines contained different terminal deletions of chromosome 5. An analysis of the breakpoints of these deletions has demonstrated that the DHFR gene resides in the q11----q22 region.     

Crystal structure of human uroporphyrinogen III synthase.

Uroporphyrinogen III synthase, U3S, the fourth enzyme in the porphyrin biosynthetic pathway, catalyzes cyclization of the linear tetrapyrrole, hydroxymethylbilane, to the macrocyclic uroporphyrino gen III, which is used in several different pathways to form heme, siroheme, chlorophyll, F(430) and vitamin B(12). U3S activity is essential in all organisms, and decreased activity in humans leads to the autosomal recessive disorder congenital erythropoetic porphyria. We have determined the crystal structure of recombinant human U3S at 1.85 A resolution. The protein folds into two alpha/beta domains connected by a beta-ladder. The active site appears to be located between the domains, and variations in relative domain positions observed between crystallographically independent molecules indicates the presence of flexibility that may be important in the catalytic cycle. Possible mechanisms of catalysis were probed by mutating each of the four invariant residues in the protein that have titratable side chains. Additionally, six other highly conserved and titratable side chains were also mutated. In no case, however, did one of these mutations abolish enzyme activity, suggesting that the mechanism does not require acid/base catalysis.     

Cystathionine beta synthase: gene dosage effect in trisomy 21

The enzymatic activity of cystathionine beta synthase has been studied in fibroblasts of nine patients with regular trisomy 21. An excess of CBS activity was found in trisomy 21 with a trisomy 21/normal ratio equal to 1.66. A 1.04 ratio was found in 21q21----21 p ter monosomy; a 1.04 and 0.99 ratio was found in two 21 qter----21q22.3 monosomies; a 1.14 ratio in 21 qter----21q22 monosomy; a 0.89 ratio in a 21q21----21 pter trisomy; an excess of CBS activity was found in a 21q22.1 ----21q21 trisomy with a 1.57 ratio. These results show a gene dosage effect in human fibroblasts trisomic for chromosome 21 and suggest the assignment of human CBS locus between 21q22.1 and 21q21.     

Phylogenetic analysis of uroporphyrinogen III synthase (UROS) gene

The uroporphyrinogen III synthase (UROS) enzyme (also known as hydroxymethylbilane hydrolyase) catalyzes the cyclization of hydroxymethylbilane to uroporphyrinogen III during heme biosynthesis. A deficiency of this enzyme is associated with the very rare Gunther''s disease or congenital erythropoietic porphyria, an autosomal recessive inborn error of metabolism. The current study investigated the possible role of UROS (Homo sapiens [EC: 4.2.1.75; 265 aa; 1371 bp mRNA; Entrez Pubmed ref {"type":"entrez-protein","attrs":{"text":"NP_000366.1","term_id":"4557873","term_text":"NP_000366.1"}}NP_000366.1, {"type":"entrez-nucleotide","attrs":{"text":"NM_000375.2","term_id":"171543867","term_text":"NM_000375.2"}}NM_000375.2]) in evolution by studying the phylogenetic relationship and divergence of this gene using computational methods. The UROS protein sequences from various taxa were retrieved from GenBank database and were compared using Clustal-W (multiple sequence alignment) with defaults and a first-pass phylogenetic tree was built using neighbor-joining method as in DELTA BLAST 2.2.27+ version. A total of 163 BLAST hits were found for the uroporphyrinogen III synthase query sequence and these hits showed putative conserved domain, HemD superfamily (as on 14^th Nov 2012). We then narrowed down the search by manually deleting the proteins which were not UROS sequences and sequences belonging to phyla other than Chordata were deleted. A repeat phylogenetic analysis of 39 taxa was performed using PhyML and TreeDyn software to confirm that UROS is a highly conserved protein with approximately 85% conserved sequences in almost all chordate taxons emphasizing its importance in heme synthesis.     

Assignment of the gene locus for human phosphoglucomutase 3 to chromosome 6q12-qter

Segregation of human PGM3 has been analyzed in somatic cell hybrids between mouse A9 cells and human fibroblasts carrying a reciprocal translocation: 46,XX, t(6;7) (q12;p14). The enzyme marker segregates with the 7p+ chromosome indicating that the PGM3 gene is located on 6q12 greater than qter.     

Assignment of the bovine uridine monophosphate synthase gene to the bovine Chromosome region 1q34-36 by FISH

The BUMPS gene has been chromosomally assigned by fluorescence in situ hybridization (FISH), combined with plotting of the resulting signals in histograms. In three experiments a peak on Chromosome (Chr) 1q34-36 could be observed.     

Assignment of the αB-crystallin gene to human chromosome 11

Using a human αB-crystallin genomic probe and human-mouse somatic cell hybrids, the human αB-gene was assigned to chromosome 11 and further corroborated by in situ hybridization to normal metaphase chromosomes. This assignment confirmed and regionally mapped the locus to q22.3–23.1.     

Assignment of the gene for neuroendocrine protein 7B2 (SGNE1 locus) to mouse chromosome region 2[E3-F3] and to human chromosome region 15q11-q15

The gene for 7B2, a protein found in the secretory granules of neural and endocrine cells (gene symbol SGNE1) was localized to the E3-F3 region of mouse chromosome 2 and to the q11-q15 region of human chromosome 15. This was determined by in situ hybridization, using a mouse 7B2 cDNA and an intronic fragment of the corresponding human gene as probes. The respective locations of SGNE1 in the two species correlate with the conservation of loci between these subregions of mouse chromosome 2 and human chromosome 15. Clinically, the human SGNE1 DNA fragment may serve as a molecular probe of this locus in both the Prader-Willi and the Angelman syndromes, which are often accompanied by submicroscopic chromosomal deletions in the 15q11-15q13 region.     

Purification and properties of uroporphyrinogen III synthase from human erythrocytes

Uroporphyrinogen III synthase (hydroxymethylbilane hydro-lyase (cyclizing); EC 4.2.1.75), the fourth enzyme in the heme biosynthetic pathway, was purified to homogeneity from human erythrocytes. For enzyme purification and characterization, a sensitive coupled enzyme assay was used which generated the substrate, hydroxymethylbilane; the oxidized product, uroporphyrin III, was quantitated by high pressure liquid chromatography. Uroporphyrinogen III synthase was initially separated from delta-aminolevulinate dehydratase and hydroxymethylbilane synthase by a preparative anion exchange chromatographic step. Subsequent chromatography on hydroxyapatite, phenyl-Sepharose, and Sephadex G-100 purified the enzyme about 70,000-fold with an 8% yield. Homogeneous enzyme was obtained following a final C4-reversed phase high pressure liquid chromatographic step which removed a single major and several minor protein contaminants from the enzyme. The purified enzyme had a specific activity of over 300,000 units/mg, an isoelectric point of 5.5, and was thermolabile (t1/2 at 60 degrees C approximately 1 min). Molecular weight studies by gel filtration (Mr approximately equal to 30,000) and analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr approximately equal to 29,500) were consistent with the enzyme being a monomer. Using hydroxymethylbilane as substrate, the purified enzyme formed uroporphyrinogen III in the absence of hydroxymethylbilane synthase or other cofactors. The pH optimum was 7.4 and the Km for hydroxymethylbilane was 5-20 microM. The enzyme was activated by Na+, K+, Mg+, and Ca2+ and was inhibited by Cd2+, Cu2+, Hg2+, and Zn2+. Amino acid composition analysis was performed, and the N-terminal sequence, Met-Lys-Val-Leu-Leu-Leu, was determined by microsequencing. The availability of the purified enzyme should permit investigation of its reaction mechanism as well as facilitate biochemical and molecular studies of the genetic defect in congenital erythropoietic porphyria.     

Random decarboxylation of uroporphyrinogen III by human hepatic uroporphyrinogen decarboxylase

The type III heptacarboxylic porphyrinogens derived from enzymic decarboxylation of an acetic acid substituent on uroporphyrinogen III to a methyl group by human hepatic uroporphyrinogen decarboxylase has been analysed by reversed-phase high-performance liquid chromatography with electrochemical detection. The results showed that all four possible heptacarboxylic acid porphyrinogen isomers, with the methyl group attached to rings A, B, C and D of the tetrapyrrole macrocycle, respectively, were formed in almost equal proportions. It was concluded that the normal pathway of uroporphyrinogen III decarboxylation in human liver follows a random mechanism.