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Identification of a z-Crystallin (Quinone Reductase)-like 1 Gene(CRYZL1) Mapped to Human Chromosome 21q22.1

Min-Young Kim, Hye-Kyung Lee, Jung-Sun Park, Sun-Hwa Park,*Hyuk-Bang Kwon, and Jaemog Soh1

Hormone Research Center and Department of Biology, College of Natural Sciences, Chonnam National University, Kwangju, 500-757;and *Department of Anatomy, College of Medicine, Korea University, Seoul, 136-705, South Korea

Received September 29, 1998; accepted December 10, 1998

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To identify a new gene(s) located on the yeast arti-cial chromosome (YAC) clone D142H8 that wasapped to human chromosome 21q22.1, purified YACNA from the clone was utilized directly as a probe to

creen a human brain cDNA library after the suppres-ion of human repetitive DNA. One cDNA clone hy-ridizing specifically to the YAC D142H8 DNA wasdentified. The clone has an insert of 1341 bp and theongest open reading frame of 349 amino acids. Aearch of GenBank revealed that the clone has a highegree of homology to z-crystallin (quinone reductase)t the amino acid level, and its nucleotide sequenceepresents the expressed sequence from the 50-kb seg-ent of the human chromosome 21q11.1. Thus a new

ene was named CRYZL1 (z-crystalline-like 1).enomic Southern blot with total human and yeastNAs suggests that CRYZL1 might be a single-copyene. The fluorescence in situ hybridization proce-ure was applied, and the results showed that theene mapped to the human chromosome 21q22.1 sub-and. The CRYZL1 mRNA was expressed in heart,rain, skeletal muscle, kidney, pancreas, liver, and

ungs but at different levels in different tissues. © 1999

cademic Press

Human chromosomes are known to contain largeumbers of genes ranging from 60,000 to 80,000 (5).he identification and mapping of all human genes asell as the determination of the nucleotide sequence ofbillion bp of the human haploid genome are the final

oal of the Human Genome Project. To accomplish thisoal, the application of multiple approaches is neededo identify new genes on the human chromosomes.andom sequencing of the ESTs (expressed sequence

ags) from different human tissue cDNA libraries has

Sequence data from this article have been deposited with theMBL/GenBank Data Libraries under Accession No. AF029689.

1 To whom correspondence should be addressed. Fax: 062-530-409. E-mail: jmsoh@chonnam.chonnam.ac.kr.

enomics 57, 156–159 (1999)rticle ID geno.1998.5714

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888-7543/99 $30.00opyright © 1999 by Academic Pressll rights of reproduction in any form reserved.

ew genes and facilitating the generation of the tran-cript map of the chromosomes (7). The exon-trappingrocedure has been a powerful technique in the isola-ion of exons from the cloned genomic DNA, which inurn can be used for the identification of new genes (1).

relatively straightforward way of identifying a newene is to use the genomic DNA fragments cloned inosmid or lambda vectors as hybridization probes forcreening the corresponding cDNA (2). The use of aarge DNA fragment cloned into YAC2 vectors as hy-ridization probes has also provided an alternativeoute to identify uncharacterized genes (3). The YAC142H8 used in this study contains the genes encodinghosphoribosylglycinamide formyltransferase (GART)nd interferon-g receptor b (IFN-gRb), also known asccessory factor-1 (AF-1) of the human interferon re-eptor-g (11, 12).YAC D142H8 was obtained from Dr. D. Patterson

Eleanor Roosevelt Institute, Denver, CO). The YAC142H8 DNA (540 kb) was purified by excision of theel slices containing the YAC DNA following electro-horesis on pulsed-field gel electrophoresis (PFGE)170 V for 24 h at 15°C with a pulse time of 70 s). Theuman brain cDNA library (Clontech HL1128a, cere-ellum, 59-Stretch cDNA) was screened with purifiedAC D142H8 after the suppression of signals fromighly repetitive human DNA sequence and the YACector sequence (3). Seventy-five nanograms of theAC DNA was labeled by the random priming proce-ure (4) to a specific activity of 5 3 109 cpm/mg, and theabeled probe was denatured by being boiled for 5 minn the presence of 100 mg of human Cot-1 DNA (BRL5279-011) and 20 mg of sheared pBR322 plasmid in a00-ml volume (3). Then the denatured DNAs werellowed to reassociate to a Cot value of approximately

2 Abbreviations used: YAC, yeast artificial chromosome; GART,hosphoribosylglycinamide formyltransferase; IFN-gRb, interfer-n-g receptor b, FISH: fluorescence in situ hybridization; ORF, openeading frame; CRYZL1, z-crystallin-like 1.

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0 in 63 SSC at 68°C (10) and used for the probe. Thelots were preincubated for 4 h with the prehybridiza-ion solution and hybridized with the suppressed probeor 20 h. After the membrane was washed with 23SC, 0.1% SDS at RT for 30 min twice and with 0.13SC, 0.1% SDS at 65°C for 30 min twice, the blots werexposed to X-ray film. The positive clones were puri-ed as described above with secondary and tertiarycreening.To identify new genes located on the YAC clone142H8, the purified YAC DNA from the clone wastilized directly as a probe to screen a human brainDNA library after the suppression of human repet-tive DNA as described in a previous report (3). Ini-ially five positive clones were identified out of aillion plaques and subsequent analysis revealed

hat only one clone (lMK4) hybridized specifically tohe YAC DNA sequence and was free of highly repet-tive human sequences. The insert from lMK4 wasloned into pBluescript, which is referred to asMK4. The determination of the nucleotide sequencef the plasmid pMK4 revealed that it has 1341 nt ofnsert and contains a poly(dA) tract at the 39-termi-us. It generates the largest open reading frameORF) of 349 amino acids (GenBank Accession No.F029689). A comparison of the deduced amino acidequence of pMK4 against the protein database re-ealed that it has a high degree of similarity withuman z-crystallin (quinone reductase) (6) as shown

n Fig. 1. The data revealed that it has 27.7% se-uence identity and 49.1% similarity with human-crystallin (quinone reductase) within the regionrom aa 25 to 183, which suggests that the pMK4lone might code for a z-crystallin-like protein. Thuse have named this gene CRYZL1 (z-crystallin-like). The conserved NAD(P)H binding site, which isxGxxAxxA (8) is located at aa 155, although therst amino acid was alanine instead of glycine as

FIG. 1. Comparison of the amino acid sequence of CRYZL1 withhose of human z-crystallin (quinone reductase). BlastX was carriedut, and the results showed that the CRYZL1 sequence has 27.7%equence identity and 49.1% similarity with human z-crystallinithin a 159-amino-acid sequence from 25 to 183. The conservedAD(P)H binding site is marked with an asterisk. Letters between

wo lines represent identical sequences, and a plus sign representsonserved sequences.

mino acid sequence of CRYZL1 also has a statisti-ally significant degree of similarity with trans-2-noyl-CoA reductase (crotonyl-CoA reductase) (P 5.824) (14), which is a member of the quinone oxi-oreductase superfamily. The amino acid sequence ofRYZL1 from 98 to 224 also has a high degree ofomology with the enoyl reductase domain (1619 –745) of human fatty acid systhase (9) (P 5 8.8 3027) (data not shown).A search of GenBank with the nucleotide sequence

evealed that the CRYZL1 cDNA sequence representshe expressed sequence from the 50,188-bp genomicNA segment of the human chromosome 21q11.1

GenBank Accession No. AP000047). The nucleotideequence of the genomic DNA from 11,423 to 46,566ay contain 11 exons of the CRYZL1 gene. However,

he nucleotide sequence of the CRYZL1 cDNA se-uence from 1 to 121 is not included in the genomicequence, which suggests that putative exon I and the9-upstream sequence of the CRYZL1 gene are missingn the genomic DNA sequence. Taken together, theata indicate that genomic structure of the CRYZL1ene spans more than 35 kb.A genomic Southern blot analysis was carried outith total human and yeast DNAs as shown in Fig. 2A.he data indicated that the CRYZL1 gene might be aingle-copy gene. In addition, the results showed thatcoRI and XbaI fragments from the YAC clone match

hose from human DNA. The location of the CRYZL1ene on the YAC D142H8 with respect to two NotI sitesas determined. Genomic Southern blot analysis

howed that CRYZL1 and AF-1 (IFN-gRb) genes areocated on the 140-kb centric NotI fragment while theART gene is mapped on the middle NotI fragment

Fig. 2D; Ref. 13; data not shown).Since the CRYZL1 cDNA was isolated with YAC142H8, which has been mapped to human chromo-

ome 21q22.1, the location of the gene should be theame as that of the YAC. To confirm that the CRYZL1ene was located in the subband, a l genomic clonel4PG5) corresponding to the CRYZL1 cDNA was iso-ated and used for the probe for FISH analysis. Theesults showed that CRYZL1 is mapped to humanhromosome 21q22.1 as shown in Fig. 2B.A Northern tissue blot analysis was carried out to

etermine the tissue distribution of the CRYZL1RNA. As shown in Fig. 2C, the CRYZL1 mRNA was

bout 1.4 kb, the same size as the insert of pMK4,hich indicates that the clone might be a full-length

lone. The mRNA was highly expressed in the heart,he brain, and skeletal muscle and moderately ex-ressed in the placenta, kidney, and pancreas. Theonger exposure revealed that the liver and lungslso retained the CRYZL1 mRNA. Therefore it wasoncluded that the expression of the CRYZL1 geneight be ubiquitous.

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ACKNOWLEDGMENTS

This work was supported by KOSEF (961-0503-024-2), Hormoneesearch Center (HRC-97-0201), and Chonnam National Uni-ersity.

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YAC D142H8 DNAs. The Southern blot was made and then probedan placenta and the YAC chromosomal DNAs, respectively. (B)l4PG5 was used for FISH analysis. The slides were counterstaninedhuman chromosome 21q22.1 as indicated by the arrows. (C) Tissue7760-1) was hybridized with 32P-labeled CRYZL1 cDNA. The longer(D) Location of the CRYZL1 gene on the YAC D142H8. The CRYZL1ent.

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