Perfect Conservation Research Articles

Comparative analysis of mouse hepcidin 1 and 2 genes: evidence for different patterns of expression and co-inducibility during iron overload

In contrast to the human genome, the mouse genome contains two HEPC genes encoding hepcidin, a key regulator of iron homeostasis. Here we report a comparative analysis of sequence, genomic structure, expression and iron regulation of mouse HEPC genes. The predicted processed 25 amino acid hepcidin 2 peptide share 68% identity with hepcidin 1 with perfect conservation of eight cysteine residues. Both HEPC1 and HEPC2 genes have similar genomic organization and have probably arisen from a recent duplication of chromosome 7 region, including the HEPC ancestral gene and a part of the adjacent USF2 gene. Insertion of a retroviral intracisternal A-particle element was found upstream of the HEPC1 gene. Both genes are highly expressed in the liver and to a much lesser extent in the heart. In contrast to HEPC1, a high amount of HEPC2 transcripts was detected in the pancreas. Expression of both genes was increased in the liver during carbonyl–iron and iron–dextran overload. Overall our data suggest that both HEPC1 and HEPC2 genes are involved in iron metabolism regulation but could exhibit different activities and/or play distinct roles.

Reductive genome evolution in Buchnera aphidicola.

We have sequenced the genome of the intracellular symbiont Buchnera aphidicola from the aphid Baizongia pistacea. This strain diverged 80-150 million years ago from the common ancestor of two previously sequenced Buchnera strains. Here, a field-collected, nonclonal sample of insects was used as source material for laboratory procedures. As a consequence, the genome assembly unveiled intrapopulational variation, consisting of approximately 1,200 polymorphic sites. Comparison of the 618-kb (kbp) genome with the two other Buchnera genomes revealed a nearly perfect gene-order conservation, indicating that the onset of genomic stasis coincided closely with establishment of the symbiosis with aphids, approximately 200 million years ago. Extensive genome reduction also predates the synchronous diversification of Buchnera and its host; but, at a slower rate, gene loss continues among the extant lineages. A computational study of protein folding predicts that proteins in Buchnera, as well as proteins of other intracellular bacteria, are generally characterized by smaller folding efficiency compared with proteins of free living bacteria. These and other degenerative genomic features are discussed in light of compensatory processes and theoretical predictions on the long-term evolutionary fate of symbionts like Buchnera.

Cloning of cDNA encoding the nuclear form of chicken sterol response element binding protein-2 (SREBP-2), chromosomal localization, and tissue expression of chicken SREBP-1 and -2 genes

Sterol regulatory element binding protein-1 and -2 (SREBP-1 and -2) are key transcription factors involved in the biosynthesis of cholesterol and fatty adds. The SREBP have mainly been studied in rodents in which lipogenesis is regulated in both liver and adipose tissue. There is, however, a paucity of information on birds, in which lipogenesis occurs essentially in the liver as in humans. As a prelude to the investigation of the role of SREBP in lipid metabolism regulation in chicken, we sequenced the cDNA, encoding the mature nuclear form of chicken SREBP-2 protein, mapped SREBP-1 and -2 genes and studied their tissue expressions. The predicted chicken SREBP-2 amino acid sequence shows a 77 to 79% identity with human, mouse, and hamster homologues, with a nearly perfect conservation in all the important functional motifs, basic, helix-loop-helix, and leucine zipper (bHLH-Zip) region as well as cleavage sites. As in the human genome, SREBP-1 and SREBP-2 chicken genes are located on two separate chromosomes, respectively microchromosome 14 and macrochromosome 1. Tissue expression data show that SREBP-1 and SREBP-2 are expressed in a wide variety of tissues in chicken. However, unlike SREBP-2, SREBP-1 is expressed preferentially in the liver and uropygial gland, suggesting an important role of SREBP-1 in the regulation of lipogenesis in avian species.

Cork quality estimation by using Compton tomography

The quality control of cork stoppers is mandatory in order to guarantee the perfect conservation of the wine. Several techniques have been developed but until now the quality control was essentially related to the status of the external surface. Thus possible cracks or holes inside the stopper will be hidden. In this paper a new technique based on X-ray Compton tomography is described. It is a non-destructive technique that allows one to reconstruct and visualize the cross-section of the cork stopper analyzed, and so to put in evidence the presence of internal imperfections. Some results are reported and compared with visual classification.

Cloning of the murine non-muscle myosin heavy chain IIA gene ortholog of human MYH9 responsible for May-Hegglin, Sebastian, Fechtner, and Epstein syndromes

Mutations in the non-muscle myosin heavy chain IIA gene ( MYH9) are responsible for May-Hegglin anomaly, Sebastian, Fechtner and Epstein syndromes. These ‘ MYH9-related’ diseases are inherited as an autosomal dominant trait and are characterized by a variable expressivity of clinical features, including macrothrombocytopenia, deafness, nephrites, cataract, and Döhle-like leukocyte inclusions. To gain information of the function of the non-muscle myosin heavy chain IIA protein (NMMHC-IIA), we have identified the murine orthologue Myh9 gene. The gene is localized in a region of chromosome 15 and encodes a predicted protein of 1960 amino acids. This protein shows a high homology to the human NMMHC-IIA with 98% identity. The Myh9 exon-intron junctions were deduced from a murine genomic clone that revealed a perfect conservation of the exon structure between the human and mouse gene. Myh9 is expressed in liver, kidney, lung, and spleen. A low level of transcripts was detected also in heart and brain while no expression was revealed in skeletal muscle and testis. In vertebrates, NMMHC-IIA shows a striking degree of homology to NMMHC-IIB, which is expressed at higher level in mouse brain and testis than in other tissues, confirming the hypothesis that the two non-muscle myosins have different functional roles within cells.

Isolation and Characterization of a Calcium Channel Gene, Cacna1f, the Murine Orthologue of the Gene for Incomplete X-Linked Congenital Stationary Night Blindness

The mutant L-type calcium channel α1-subunit gene, CACNA1F, was recently identified as the gene responsible for incomplete X-linked congenital stationary night blindness. The 6070-bp mRNA transcript is predicted to encode a 1977-amino-acid pore-forming protein with cytoplasmic amino- and carboxyl-termini separated by four homologous repeat domains, each consisting of six transmembrane segments. CACNA1F has been shown to be preferentially expressed in the retina, indicative of a specific functional role in visual processing. We have established the complete sequence of the murine orthologue of CACNA1F, namely Cacna1f. The total length of the mRNA transcript of the murine gene was established to be 6080 bp with an open reading frame that translates into a 1985-amino-acid protein. Cacna1f is highly homologous to the human sequence, with 90% identity at the amino acid level and almost perfect conservation between the functional domains. Furthermore, as in the human gene, the 3′ end of the Cacna1f gene maps within 5 kb of the 5′ end of the mouse synaptophysin gene in a region orthologous to Xp11.23. Using in situ hybridization, Cacna1f was found to be expressed in the inner and outer nuclear layers and the ganglion cell layer of the retina.

The diversity of cadherins and implications for a synaptic adhesive code in the CNS.

There are now, conservatively, at least 80 cadherins likely to be expressed in nervous tissue. These can be grouped into two broad categories: the classical cadherins and the CNR/protocadherins. There is already data showing that certain protocadherins are homophilically adhesive (Obata et al. 1995xObata, S., Sago, H., Mori, N., Rochelle, J.M., Seldin, M.F., Davidson, M., St. John, T., Taketani, S., and Suzuki, S.T. J. Cell Sci. 1995; 108: 3765–3773PubMedSee all ReferencesObata et al. 1995) and have a synaptic localization. Whether this is the full extent of their in vivo binding preference remains unclear. One of the notable structural features of the newly described protocadherins is an RGD (Arg-Gly-Asp) sequence conserved in the amino-terminal domain. This suggests the possibility that the protocadherins may also act as membrane-associated ligands for integrins. We have performed secondary structure modeling of some of these protocadherins, and the RGD sequence is predicted to protrude as a loop between two secondary structure elements, just as do functional RGD sequences from fibronectins (Shapiro and Colman 1998xShapiro, L. and Colman, D.R. Curr. Opin. Neurobiol. 1998; 8: 593–599Crossref | PubMed | Scopus (32)See all ReferencesShapiro and Colman 1998). Integrins are expressed early in neurogenesis and one—at least—is localized to synapses (Einheber et al. 1996xEinheber, S., Schnapp, L.M., Salzer, J.L., Cappiello, Z.B., and Milner, T.A. J. Comp. Neurol. 1996; 370: 105–134Crossref | PubMed | Scopus (98)See all ReferencesEinheber et al. 1996), but their partners are unknown. The precedent exists for integrin binding of transmembrane proteins (ICAM and VCAM) to function in cell–cell (rather than cell–matrix) adhesion. Also, a Drosophila learning mutant, volado, has been traced to an integrin mutation (Grotewiel et al. 1998xGrotewiel, M.S., Beck, C.D., Wu, K.H., Zhu, X.R., and Davis, R.L. Nature. 1998; 391: 455–460Crossref | PubMed | Scopus (221)See all ReferencesGrotewiel et al. 1998), suggesting that this mutation may have direct synaptic effects.What might be the functional implications of the remarkable genomic organization of the protocadherins uncovered by Wu and Maniatis? As suggested above, consolidation of these protocadherin genes in one region of a chromosome provides the potential for a central control mechanism for their expression. In this arrangement, the ectodomains are evolving their specificities separately from the cytoplasmic domain, which remains true to its common function for all ectodomains to which it is joined. The nearly perfect conservation of the constant cytoplasmic exons between mouse and human is evidence for evolutionary pressure to keep this domain very stable. In a conventional gene arrangement, a large number of complete genes would be needed to encode the different extracellular adhesive specificities, and each gene would of course contain those exons encoding the conserved cytoplasmic domain. The common cytoplasmic elements would be represented, then, multiple times throughout the genome. The novel and tightly integrated arrangement of the newly described protocadherin locus “streamlines” the conventional gene organization and may minimize potentially disadvantageous homologous recombination. It will be of great interest to identify all the intracellular binding partners of these conserved cytoplasmic segments, since they differ so markedly from their catenin-binding counterparts in the classical cadherins.Although there may be several adhesion mechanisms operative at the synapse that almost certainly involve members of other adhesion protein families, it is now clear that our broad knowledge of the cadherins provides a springboard for framing new and reframing old problems of profound importance to molecular neuroscience. Will topographic patterns of different adhesive molecules be found that delineate neural systems, like the olfactory and retinotectal systems? Will we find that the identity of synaptic adhesion molecules correlates with the type of neurotransmitter receptor at a particular synapse? Do morphogen gradients progressively restrict or activate the set of adhesion molecules that are expressed on the neurite surface as the target nears? How many specifiers of adhesivity are expressed on a single neuron's surface, and what is the distribution pattern of these adhesion molecules with respect to the thousands of synapses this neuron may form? Answers to these and related questions must yield a new and integrated view of synaptic adhesion and synaptic function, and the factors affecting their interrelationship, essential for a complete understanding of the molecular mechanisms underlying both the formation of nerve connections and the physiology of the mature synapse.‡To whom correspondence should be addressed (e-mail: colman@msvax.mssm.edu and shaprio@anguilla.physbio.mssm.edu).

Transverse-junction-stripe GaAs-AlGaAs lasers for squeezed light generation

We fabricated 850-nm GaAs transverse-junction-stripe (TJS) lasers by an improved metal-organic chemical vapor deposition (MOCVD) and double Zn diffusion process. The high V/III ratio used during the MOCVD growth significantly reduced the intermixing of GaAs active layer and AlGaAs cladding layers. The modified process realized good confinement for both carriers and photons and smaller saturable loss. We measured squeezing of -2.8 dB (-4.5 dB after correction for detection efficiency) at a pumping rate of I/I/sub th//spl ap/20 from these lasers, which is in close agreement with the theoretical limit. This squeezing remained unchanged under injection locking, indicating almost perfect conservation of the intensity noise correlation among the longitudinal modes. These TJS lasers had very small low-frequency 1/f noise.

Molecular Cloning of MouseLrp7(Lr3) cDNA and Chromosomal Mapping of Orthologous Genes in Mouse and Human

LRP7 (the HGMW-approved gene symbol for LR3) is a novel member of the low-density lipoprotein receptor (LDLR) family. The unique modular features in both its extracellular and its intracellular regions and functional analysis suggest it to be a multifunctional protein with mitogenic activity and potential ligand-induced signal transduction capability. We describe here the cloning ofLrp7,the orthologue of the human LRP7 gene, from a mouse liver cDNA library and its expression profile in mouse tissues and developmental stages.Lrp7is highly homologous to the human sequence, with 95% identity at the amino acid level and a perfect conservation of all the recognizable functional domains. A 6-kb mRNA transcript could be detected in all the tissues examined, with the highest expression levels in liver, heart, and lung and the lowest levels in brain and spleen. A similar expression level of a smaller transcript (4.5 kb) was seen at the four embryonic stages examined. We mapped theLrp7gene to mouse chromosome 19B and the LRP7 gene to human chromosome 11q13.4. The LRP7 gene was also localized by radiation hybrid mapping between markers D11S24270 and D11S1975.

A 1.5 kb repeat sequence flanks the suppressor of forked gene at the euchromatin-heterochromatin boundary of the Drosophila melanogaster X chromosome.

A 1.5 kilobasepair repeated DNA sequence is duplicated in direct orientation so as to flank the suppressor of forked gene in the euchromatin-heterochromatin transition region on the X chromosome of Drosophila melanogaster. These two copies are almost identical, but DNA blotting, analysis of cloned sequences and database searches show that elsewhere in the genome, homologous sequences are poorly conserved. They are often associated with other repeats, suggesting that they may belong to a scrambled and clustered middle repetitive DNA family. The sequences do not appear to be related to transposable elements and their location in different strains is conserved. In situ hybridization to metaphase chromosomes shows that homologous sequences are concentrated in the pericentric regions of the autosomes and the X chromosome. The sequences are not significantly under-represented in DNA from polytene tissue and must lie in the replicated regions of polytene chromosomes. The almost perfect conservation of the two repeats around suppressor of forked in D. melanogaster suggests they arose by duplication or gene conversion. Suppression of recombination in this chromosomal region presumably allows this unusual organization to be stably maintained. In the X-ray induced allele, suppressor of forked-L26, the sequence between the repeats, including the gene, and one copy of the repeat have been deleted.

Microstructures and optical properties in crystallized a-Si:H multi-quantum wells using excimer laser annealing

The visible light emission from KrF excimer laser crystallized a-Si:H/a-SiN x:H multi-quantum well (MQW) films at room temperature is reported. Both X-ray diffraction (XRD) and Raman scattering spectra provide evidence for the crystallization of Si layers within the MQWs after laser annealing treatment. Transmission electron micrographs (TEM) show the perfect conservation of the layered structures and proves the mechanism of constrained crystallization by stable a-SiN x :H barrier layers during laser annealing. The peak wavelength of visible PL spectra of the crystallized samples is around 640 nm (∼ 1.94 eV) and does not shift with different exposure energy. The FWHM of the PL peak becomes narrower to 0.4 eV when the irradiation energy density is increased to 0.9 J/cm 2.

Cloning and sequence analysis of the human SNAP25 cDNA

During the screening of human brain-specific cDNAs by a modified differential hybridization analysis, we found a clone which was highly and specifically expressed in the (adult) brain. This clone was subjected to nucleotide sequence analysis and proven to be a nearly full-length cDNA of a human homologue of the previously reported mouse synaptosomal-associated protein 25 (mSNAP25). The human SNAP25 (hSNAP25) and mSNAP25 showed perfect amino-acid sequence conservation.

Analysis of revertants from respiratory deficient mutants within the center N of cytochrome b in Saccharomyces cerevisiae

Four modified cytochrome b's carrying mononucleotide substitutions affecting center N residues were analysed. The mutant carrying a G33D change does not incorporate heme into the apocytochrome b and fails to grow on non-fermentable carbon sources. Out of 85 genetically independent revertants derived from this mutant, 82 were true back-mutants restoring the wild type sequence (D33G). The remaining three replaced the aspartic acid by an alanine (D33A) indicating that small size residues are best tolerated at this position which is consistent with the perfect conservation of the G33 during evolution. This glycine may be of crucial importance for helix packing around the hemes. The replacement of methionine at position 221 by lysine (M221K.) produced a non-functional cytochrome b [(1993) J. Biol. Chem. 268,15626-15632]. Non-native revertants replacing the lysine 221 by glutamic acid (K221E) or glutamine (K221Q) expressed a selective resistance to antimycin and antimycin derivatives having a modified dilactone ring moiety. Cytochrome b residues in 33 and in 221 seemed to contribute to the quinone reduction (QN) site of the cytochrome bc1 complex. Possible intramolecular interactions between the N-tenninal region and the loop connecting helices IV and V of cytochrome b are proposed.

RepFIB: A Basic Replicon of Large Plasmids

The distribution of the RepFIB replicon among a total of 20 plasmid incompatibility groups was determined using Southern blot and polymerase chain reaction analysis. The presence of the replicon was confirmed in 25 of 55 plasmids tested. The majority of plasmids carrying RepFIB are from the IncF incompatibility groups, but one plasmid from Incl and one plasmid from IncP also possess the replicon. Seven different examples of RepFIB were sequenced in the minimal replicon region to obtain 1525 bp of sequence information covering the repA gene and flanking repeat regions for comparison. An analysis of these sequences plus three sequences previously reported showed almost perfect conservation of the predicted protein sequence of RepA and of the flanking DNA repeats. DNA sequence data were analyzed using maximum parsimony techniques to describe the possible evolutionary relationships of the 10 examples of RepFIB.

Primary structure of the human heparan sulfate proteoglycan from basement membrane (HSPG2/perlecan). A chimeric molecule with multiple domains homologous to the low density lipoprotein receptor, laminin, neural cell adhesion molecules, and epidermal growth factor.

We have determined the complete nucleotide and deduced amino acid sequence of the major protein core of the human heparan sulfate proteoglycan HSPG2/perlecan of basement membranes. Eighteen overlapping cDNA clones comprise 14.35 kilobase pairs (kb) of contiguous sequence with an open reading frame of 13.2 kb. The mature protein core, without the signal peptide of 21 amino acids, has a M(r) of 466,564. This large protein is composed of multiple modules homologous to the receptor of low density lipoprotein, laminin, neural cell adhesion molecules, and epidermal growth factor. Domain I, near the amino terminus, appears unique for the proteoglycan since it shares no significant homology with any other proteins. It contains three Ser-Gly-Asp sequences that could act as attachment sites for heparan sulfate glycosaminoglycans. Domain II is highly homologous to the LDL receptor and contains four repeats with perfect conservation of all 6 consecutive cysteines. Next is domain III which shares homology to the short arm of laminin A chain and contains four cysteine-rich regions intercalated among three globular domains. Domain IV, the largest module with greater than 2000 residues, contains 21 repeats of the immunoglobulin type as found in neural cell adhesion molecule. Near the beginning of this domain, there is a stretch of 29 hydrophobic amino acids which could allow the molecule to interact with the plasma membrane. Domain V, similar to the carboxyl-terminal globular G-domain of laminin A and to the related protein merosin, contains three globular regions and four EGF-like repeats. In situ hybridization and immunoenzymatic studies show a close association of this gene product with a variety of cells involved in the assembly of basement membranes, in addition to being localized within the stromal elements of various connective tissues. Our studies show that this proteoglycan is present in all vascularized tissues and suggest that this unique molecule has evolved from the utilization of modular structures with adhesive and growth regulatory properties.

Structure and evolution of the lipase superfamily.

The lipase superfamily includes three vertebrate and three invertebrate (dipteran) proteins that show significant amino acid sequence similarity to one another. The vertebrate proteins are lipoprotein lipase (LPL), hepatic lipase (HL), and pancreatic lipase (PL). The dipteran proteins are Drosophila yolk proteins 1, 2, and 3. We review the relationships among these proteins that have been established according to gene structural relatedness and introduce our findings on the phylogenetic relationships, distance relationships, and evolutionary history of the lipase gene superfamily. Drosophila yolk proteins contain a 104 amino acid residue segment that is conserved with respect to the lipases. We have used the yolk proteins as an outgroup to root a phylogeny of the lipase family. Our phylogenetic reconstruction suggests that ancestral PL diverged earlier than HL and LPL, which share a more recent root. Human and bovine LPL are shown to be more closely related to murine LPL than to guinea pig LPL. A comparison of the distance (a measure of the number of substitutions between sequences) between mammalian and avian LPL reveals that guinea pig LPL has the largest distance from the other mammals. Human, rodent, and rabbit HL show marked divergence from one another, although they have similar relative rates of amino acid substitution when compared to human LPL as an outgroup. Human and porcine PL are not as divergent as human and rat HL, suggesting that PL is more conserved than HL. However, canine PL demonstrates an unusually rapid rate of substitution with respect to the other pancreatic lipases. The lipases share several structurally conserved features. One highly conserved sequence (Gly-Xaa-Ser-Xaa-Gly) contains the active site serine. This feature, which agrees with that found in serine esterases and proteases, is found within the entire spectrum of lipases, including the evolutionarily unrelated prokaryotic lipases. We review the location and possible activity of putative lipid binding domains. We have constructed a conservation index (CI) to display conserved structural features within the lipase gene family, a CI of 1.0 signifying perfect conservation. We have found a correlation between a high CI and the position of conserved functional structures. The putative lipid-binding domains of LPL and HL, the disulfide-bridging cysteine residues, catalytic residues, and N-linked glycosylation sites of LPL, HL, and PL all lie within regions having a CI of 0.8 or higher. A number of amino acid substitutions have been identified in familial hyperchylomicronemia which result in loss of LPL function.(ABSTRACT TRUNCATED AT 400 WORDS)

Restriction fragment length polymorphisms in plant breeding and genetics

SynopsisThe usefulness of restriction fragment length polymorphisms (RFLPs) in plant breeding and genetics is discussed, with particular emphasis on tagging genes, map-based cloning, the assessment of genetic variability and distances, and comparative genome mapping.The Department of Plant Breeding and Biometry has currently established tight linkages between RFLPs and more than 20 genes of economic importance. Approximately half of these genes confer resistance to major pathogens including nematodes, bacteria, fungi, and viruses. Other genes tagged are involved in various aspects of crop quality.Locating genes with respect to DNA markers on an RFLP map provides a starting point for cloning the genes by chromosome walking. This strategy is currently being pursued for three disease-resistance genes that have been placed on the tomato RFLP map;Pto(resistance toPseudomonas syringae),Mi(resistance to root-knot nemotodes) andTm2a(resistance to tobacco mosaic virus). Further discussion will include the construction of a yeast artificial chromosome library and the collection of additional DNA markers in the regions of interest through RAPD analysis of nearly isogenic lines.The assessment of genetic variability and fingerprinting varieties based on RFLP data will be briefly discussed.Comparative genome mapping in the family Solanaceae has allowed the relationships among tomato, potato, and pepper to be unravelled. Tomato and potato share a near perfect conservation of gene order throughout their genomes. In contrast, while pepper shares most of its single copy DNA with tomato and potato, the order of these markers is highly rearranged compared with the other two species.

Cloning and expression of cDNAs for the alpha subunit of the murine lymphocyte-Peyer's patch adhesion molecule.

cDNA clones encoding the alpha chain of the murine lymphocyte-Peyer's patch adhesion molecule (LPAM), which is associated with lymphocyte homing, have been isolated by screening with the human VLA-4 (alpha 4h) probe. Several alpha 4 antigenic determinants were identified on COS-7 cells after transfection. From overlapping clones, approximately 5 kb of contiguous nucleotide sequence have been determined, encoding a protein sequence of 1039 amino acids for the LPAM alpha chain (alpha 4m). LPAM is a member of the integrin family of cell-surface heterodimers, and alpha 4m is the murine homologue of the human alpha 4 h chain. The two proteins have a total sequence similarity of 84%, with an almost perfect conservation (31/32 amino acids) in the cytoplasmic domain. Like alpha 4h, alpha 4m is distinct from other integrin alpha chains because it has neither an I-domain nor a COOH-terminal cleavage site. The positions of the characteristic Cysteine residues are conserved, and a putative protease cleavage site is located near the middle of the protein sequence. The NH2-terminal part of the protein contains seven homologous repeats, and three of them include putative divalent cation-binding sites. These sites are among the most conserved between the alpha 4m sequence and other alpha chains, and may therefore be involved in the binding of integrin alpha and beta chains. An additional cDNA clone was isolated which shares a sequence of perfect homology with the alpha 4m encoding cDNAs, but has a unique 3' poly-A end. This observation correlates with the fact that three discrete murine RNA bands are observed in Northern blot experiments using alpha 4m as a probe, whereas only two human RNA species are described for alpha 4h, indicating a higher complexity for murine than for human sequences.

Complete Nucleotide Sequence of Human Phosphoribosyl Pyrophosphate Synthetase Subunit I (PRS I) cDNA and a Comparison with Human and Rat <italic>PRPS</italic> Gene Families<xref ref-type="fn" rid="fn1"><sup>1</sup></xref>

cDNA clones for human phosphoribosyl pyrophosphate synthetase subunit I (PRS I) were isolated from a glioblastoma cell line MGC 1 cDNA library. The longest clone contained 2,075 base pairs (bp) almost covering the 2.3-kb mRNA and the base sequence of the coding region (954 bp) had a 92.0% sequence homology with that of rat PRS I cDNA. The deduced amino acid sequences were identical between human and rat PRS I. This perfect conservation has heretofore not been reported for other enzymes involved in nucleotide metabolism and glycolysis. A comparison with other isoforms of this enzyme, PRS II and PRS III, showed that the human PRS I was 79.9 and 92.2% homologous in the coding sequence and 95.3 and 94.0% in the deduced amino acid sequence to human PRS II and PRS III, respectively. The high value of the synonymous difference between PRS I and PRS II cDNAs places their time of divergence long before that of the radiation of mammals. Based on the evolutionary rate of amino acid substitution, the PRS I and II genes probably diverged about 760 million years ago.

Mouse and human retinoic acid receptor beta 2 promoters: sequence comparison and localization of retinoic acid responsiveness.

The retinoic acid receptor beta (RAR beta) gene is a member of the family of retinoic acid/thyroid hormone receptor genes, encoding retinoic acid-inducible transcription factors. To study regulation of the RAR beta gene, genomic clones containing the mouse and human retinoic acid receptor beta 2 (RAR beta 2) promoters were isolated and approximately 1.5 kb of upstream and downstream sequences relative to the transcriptional start site were completely sequenced. Both the mouse and human RAR beta 2 promoters are highly homologous around the transcription initiation site, with perfect conservation of the TATA box and retinoic acid responsive element (RARE). Promoter activation studies in P19 EC cells show, that the RARE in both the human and mouse promoters confers RA-responsiveness to the RAR beta 2 promoter. However, sequences located immediately upstream of the RARE also confer RA-inducibility to both the mouse and human RAR beta 2 gene promoters. This region contains conserved consensus sequences for a TPA-responsive element (TRE) and cAMP-responsive element (CRE), suggesting that in addition to regulation by RA receptors other transcription factors regulate RAR beta 2 expression in EC cells. Furthermore, the availability of the mouse RAR beta 2 promoter should facilitate studies for transgene expression and gene targeting experiments in embryonic stem cells.