NORMAL GENOMIC

• Nal B et al.
• Wdr12, a mouse gene encoding a novel WD-Repeat Protein with anotchless-like amino-terminal domain.
• Genomics. 2002; 79: 77-86
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• The WD-repeat protein family consists of a large group of structurallyrelated yet functionally diverse proteins found predominantly ineukaryotic cells. These factors contain several (4-16) copies of arecognizable amino-acid sequence motif (the WD unit) thought to beorganized into a "propeller-like" structure involved in protein-proteinregulatory interactions. Here, we report the cloning of a mouse cDNA,referred to as Wdr12, which encodes a novel WD-repeat protein of 423 aminoacids. The WDR12 protein was predicted to contain seven WD units and anuclear localization signal located within a protruding peptide betweenthe third and fourth WD domains. The amino-terminal region showssimilarity to that of the Notchless WD repeat protein. Sequencecomparisons revealed WDR12 orthologs in various eukaryotic species. Wdr12seems to correspond to a single-copy gene in the mouse genome, locatedwithin the C1-C2 bands of chromosome 1. These data, together with theresults of Wdr12 gene expression studies and evidence of in vitro bindingof WDR12 to the cytoplasmic domain of Notch1, led us to postulate afunction for the WDR12 protein in the modulation of Notch signalingactivity.

• Zhang H, Nichols K, Thorgaard GH, Ristow SS
• Identification, mapping, and genomic structural analysis of animmunoreceptor tyrosine-based inhibition motif-bearing C-type lectin fromhomozygous clones of rainbow trout (Oncorhynchus mykiss).
• Immunogenetics. 2001; 53: 751-9
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• Utilizing a spleen-derived cDNA library and rapid amplification of cDNA 5'ends, we cloned a novel type II C-type lectin from two homozygous clonesof rainbow trout. The cDNA is 2535 bp in length, and contains a 1017-bpopen reading frame. From this sequence, a protein containing 339 aminoacids (aa) was deduced. Using PSI-BLAST to search the GenBank database,the deduced protein is a C-type lectin, belonging to the type II membranereceptors. The protein contains four domains: an 87-aa N-terminalcytoplasmic domain, a 21-aa transmembrane domain, an 82-aa neck domain,and a 149-aa C-terminal C-type lectin domain. Two immunoreceptortyrosine-based inhibition motifs (ITIMs) were located in the N-terminalcytoplasmic domain. RT-PCR results indicated that this gene is transcribedmainly in peripheral blood lymphocytes, spleen, kidney, and gill, and itsexpression in liver and intestine is weak. Monoclonal antibody 1.14 wasused to isolate B cells from peripheral blood lymphocytes. Analysisrevealed that this gene is highly expressed in B cells. Genomic DNA wasamplified with long-template PCR and sequenced. The gDNA is 12.0 kb inlength and contains nine exons and eight introns. The first intron of thegenes from the OSU and AR clones differed in length. Based on thisdifference, the genotype of 69 doubled-haploid offspring of OSU and ARwere screened. Subsequently, this gene was mapped on the rainbow troutlinkage map to group XXI. Results of a Southern blot indicated that thegene ( TCL-2) exists as a single copy in the rainbow trout genome. Thegenomic structure, the deduced protein structure, the tissue expressionpattern, as well as the phylogenetic analysis of the carbohydraterecognition domain based on the deduced amino acid sequence indicate thatTCL-2 resembles CD72; however, the carbohydrate recognition domainsequences of TCL-2 and CD72 are highly diverged.

• Golderer G, Werner ER, Heufler C, Strohmaier W, Grobner P, Werner-Felmayer G
• GTP cyclohydrolase I mRNA: novel splice variants in the slime mouldPhysarum polycephalum and in human monocytes (THP-1) indicate conservationof mRNA processing.
• Biochem J. 2001; 355: 499-507
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• GTP cyclohydrolase I (EC 3.5.4.16) is the first enzyme in the biosynthesisof tetrahydrobiopterin [(6R)-5,6,7,8-tetrahydro-L-biopterin,H(4)-biopterin] in mammals and of folic acid in bacteria. Here we havecharacterized the GTP cyclohydrolase I gene structure and two mRNA speciesfrom Physarum polycephalum, an acellular slime mould that synthesizesH(4)-biopterin and metabolites of the folic acid biosynthetic pathway. ItsGTP cyclohydrolase I gene consists of seven exons, and the two GTPcyclohydrolase I cDNA species isolated from Physarum encode for proteinswith 228 (25.7 kDa) and 195 (22.1 kDa) amino acids. Furthermore, weidentified two previously undescribed mRNA species ininterferon-gamma-treated human myelomonocytoma cells (THP-1) in additionto the cDNA coding for the fully functional 250-residue (27.9 kDa)protein, which is identical with that in human phaeochromocytoma cells.One of the new splice variants codes for a 233-residue (25.7 kDa) protein,whereas the other codes for the full-length protein but is alternativelyspliced within the 3'-untranslated region. In heterologous expression, theshorter proteins of Physarum as well as of THP-1 cells identified here aredegraded by proteolysis. Accordingly, only the 27.9 kDa protein wasdetectable in Western blots from THP-1 cell extracts. Quantification ofGTP cyclohydrolase I mRNA species in different human cell types with andwithout cytokine treatment showed that in addition to the correct mRNA thetwo splice variants isolated here, as well as the two splice variantsknown from human liver, are strongly induced by cytokines in cell typeswith inducible GTP cyclohydrolase I (THP-1, dermal fibroblasts), but notin cell types with constitutive GTP cyclohydrolase I expression (SK-N-SH,Hep-G2). As in human liver, splicing of the new mRNA variant found inTHP-1 cells occurs at the boundary of exons 5 and 6. Strikingly, the195-residue protein from Physarum is alternatively spliced at a homologousposition, i.e. at the boundary of exons 6 and 7. Thus alternative splicingof GTP cyclohydrolase I at this position occurs in two species highlydistant from each other in terms of evolution. It remains to be seenwhether variant proteins encoded by alternatively spliced GTPcyclohydrolase I mRNA transcripts do occur in vivo and whether theyparticipate in regulation of enzyme activity.

• Iwaki D, Osaki T, Mizunoe Y, Wai SN, Iwanaga S, Kawabata S
• Functional and structural diversities of C-reactive proteins present inhorseshoe crab hemolymph plasma.
• Eur J Biochem. 1999; 264: 314-26
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• Limulin, a sialic-acid-binding and phosphorylethanolamine-bindinghemagglutinin in the hemolymph plasma of the American horseshoe crab(Limulus polyphemus), is a hemolytic C-reactive protein [Armstrong, P.B.,Swarnakar, S., Srimal, S., Misquith, S., Hahn, E.A., Aimes, R. T. &Quigley, J.P. (1996) J. Biol. Chem. 271, 14717-14721]. We have nowidentified three types of C-reactive protein in the plasma of the Japanesehorseshoe crab (Tachypleus tridentatus), based on different affinitiesagainst fetuin-agarose and phosphorylethanolamine-agarose determined byquantitative precipitin assays using fetuin and an artificialphosphorylethanolamine-protein conjugate. Partial amino acid sequences ofthe isolated C-reactive proteins identified homologous proteins which werenamed Tachypleus tridentatus CRP-1 (tCRP-1), tCRP-2 and tCRP-3, each ofwhich possibly constitute isoprotein mixtures. tCRP-2 and tCRP-3, but nottCRP-1, agglutinated mammalian erythrocytes. tCRP-1, the most abundantC-reative protein in the plasma, exhibited the highest affinity to thephosphorylethanolamine-protein conjugate but lacked bothsialic-acid-binding and hemolytic activities. tCRP-2 bound to bothfetuin-agarose and phosphorylethanolamine-agarose, and exhibitedCa2+-dependent hemolytic and sialic-acid-binding activities, suggestive oflimulin-like properties. Furthermore, tCRP-2 exhibited a higher affinityto colominic acid, a bacterial polysialic acid. By contrast, tCRP-3 showsstronger hemolytic, sialic-acid-binding and hemagglutinating activitiesthan tCRP-2. tCRP-3 has no affinity to phosphorylethanolamine-agarose,phosphorylethanolamine-protein conjugate and colominic acid. This suggeststCRP-3 is a novel hemolytic C-reactive protein lacking a commoncharacteristic of phosphorylethanolamine-agarose binding affinity.Twenty-two clones of tCRPs with different deduced amino acid sequenceswere obtained by PCR using oligonucleotide primers based on the N-terminaland C-terminal sequences of tCRPs and with templates including genomic DNAand cDNA of hemocytes or hepatopancreas derived from one individual. Thetranslation products of the tCRP clones possess high molecular diversitywhich falls into three related groups, consistent with classificationbased on their biological activities. Only tCRP-3 contained a uniquehydrophobic nonapeptide sequence that appears in the transmembrane domainof a major histocompatibility complex class I heavy chain of rainbowtrout, suggesting the importance of the hydrophobic patch to the hemolyticactivity of tCRP-3. The structural and functional diversities of tCRPsprovide a good model for studying the properties of innate immunity ininvertebrates, which survive without the benefit of acquired immunity.

• Kruse L, Meyer G, Hildebrandt A
• A highly conserved repetitive sequence from Physarum polycephalum containsnucleotide arrangements similar to replicator sequences.
• Biochim Biophys Acta. 1993; 1216: 129-33
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• An interspersed repetitive sequence from Physarum polycephalum has beencloned and analysed. The 394 bp sequence is highly conserved and containsseveral homopolymeric (dA)-(dT) tracts capable of forming bent DNAstructures and a 10/11 match to the yeast-ARS-consensus sequence. Therepetition frequency of the described sequence is about 3000 to 7000, anumber that would fit with the distribution of replicator segments inPhysarum.

• Miyazaki K, Hamano T, Hayashi M
• Physarum vitronectin-like protein: an Arg-Gly-Asp-dependent cell-spreadingprotein with a distinct NH2-terminal sequence.
• Exp Cell Res. 1992; 199: 106-10
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• A 70-kDa protein cross-reacted with anti-bovine vitronectin was isolatedfrom slime mold Physarum polycephalum. The NH2-terminal amino acidsequence of the protein, referred to as Physarum vitronectin-like protein,did not share any homology with those of animal vitronectins. It hadcell-spreading activity, which was specifically inhibited by anArg-Gly-Asp (RGD)-containing peptide.