Cloning, Mapping, and Characterization of a Human Homologue of the Yeast Longevity Assurance Gene LAG1

Article doi:10.1006/geno.2001.6614, available online at http://www.idealibrary.com on IDEAL

INTRODUCTION

Cloning, Mapping, and Characterization of a HumanHomologue of the Yeast Longevity Assurance Gene LAG1

Hui Pan,1,* Wen-Xin Qin,2,* Ke-Ke Huo,1 Da-Fang Wan,2 Yao Yu,1 Zhi-Gang Xu,1Qian-De Hu,1 Kerong T. Gu,1 Xiao-Mei Zhou,2 Hui-Qiu Jiang,2 Ping-Ping Zhang,2

Yi Huang,2 Yu-Yang Li,1,† and Jian-Ren Gu2,†

1State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai 200433, China2National Laboratory for Oncogenes and Related Genes, Shanghai Cancer Institute, Shanghai 200032, China

*These authors contributed equally to this work.

†To whom correspondence and reprint requests should be addressed. Fax: 86 21 65643436. E-mail: [email protected]. Fax: 86 21 64177401. E-mail: [email protected].

We have identified LASS2, a previously unknown human homologue of the yeast longevityassurance gene LAG1. The LASS2 transcript is highly expressed in liver and kidney, whichis very different from the expression of the previously identified human LAG1 homologueLAG1Hs-1. Radiation hybrid mapping studies indicated that LASS2 is located on chromosome1q11. Yeast two-hybrid screening and glutathione S-transferase pull-down assays showed thatthe LASS2 protein interacts with several membrane-associated receptors or transporters.Furthermore, LASS2 protein was able to inhibit the colony formation of human hepatomacells in vitro, which suggests that this gene may be involved in the regulation of cell growth.

Key Words: human LAG1 homologue, cDNA cloning, chromosomal mapping,two-hybrid, hepatocellular carcinoma, colony formation

tate ER-to-Golgi transport of glycosylphosphatidyl inositol-

The longevity assurance gene LAG1 was originally identifiedand cloned from the baker’s yeast Saccharomyces cerevisiae. Thegene is preferentially expressed in young cells, and is able toincrease or decrease lifespan by mutation or overexpression[1]. Several LAG1 homologues have been identified in variousorganisms [2–6]. Most of the homologue proteins share a sim-ilar profile of six or seven transmembrane helices, a basicdomain between the first two helices on the amino terminus,a Lag1 motif, and a carboxy-terminal acidic domain. In S. cere-visiae, Schizosaccharomyces pombe, Caenorhabditis elegans, andArabidopsis thaliana, at least two copies of LAG1 homologuesexist. The homologues in a same organism seem to be func-tionally redundant. For example, deletion of LAG1 in baker’syeast has no visually apparent phenotype because of the pres-ence of a functional homologue, called the LAG1 cognate(LAC1). Double deletion of LAG1 and LAC1 leads to lethal [3]or slow growth and cell wall defects [7] in different geneticbackgrounds. The human and nematode C. elegans LAG1 genescould not be isolated using yeast LAG1 as a probe, probablybecause of limited nucleic acid homology; nevertheless, theycould rescue the LAG1/LAC1-deficient rescue yeast strain [3].

The molecular mechanism by which LAG1 determines yeastlongevity is unclear at present. Recent studies have shown thatyeast Lag1p and Lac1p are ER membrane proteins and facili-

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anchored proteins (EGGAP transport) [7]. In addition, thetomato homologue of Lag1p mediates resistance to sphinga-nine-analogue mycotoxins, which are inhibitors of sphingolipidbiosynthesis. As EGGAP transport is dependent on de novosphingolipid synthesis in yeast and human, it has been pro-posed that Lag1p homologues may be involved in a salvagemechanism of sphingolipid-depleted cells [2]. A more recentstudy showed that yeast Lag1p and Lac1p may directly par-ticipate in or regulate the synthesis of ceramide, which is themain source for sphingolipids synthesis in yeast [8].

In this study, we isolated a previously unknown humanLAG1 homologue, which we have called LASS2 (Homo sapienslongevity assurance homologue 2 of yeast LAG1, also knownas SP260 and LAG1Hs-2), which had a different tissue expres-sion pattern from that of the previously reported human homo-logue LAG1Hs-1 [3]. The predicted LASS2 protein has the con-sensus Lag1 motif, a C-terminal acidic domain, and fourtransmembrane helices, but lacks the basic domain and its twoflanking transmembrane helices. These lines of evidence led usto hypothesize that LASS2 has a unique function distinct fromthat of LAG1Hs-1. Furthermore, we have identified interac-tion between LASS2 and several membrane-associated recep-tors or transporters by yeast two-hybrid screening and glu-tathione S-transferase (GST) pull-down assays. A preliminarystudy showed that LASS2 protein was able to inhibit the colony

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Articledoi:10.1006/geno.2001.6614, available online at http://www.idealibrary.com on IDEAL

FIG. 1. Nucleotide sequence of human LASS2 cDNA and its deduced amino acid sequence. In the nucleotide sequence, the 5�-RACE sequence starts at nucleotide1223 (bp 1–1223), a putative poly(A) signal (AATAAA) is identified by the letters in the box, and arrows indicate the sequence and orientation of 5�-RACE primers.In the amino acid sequence, the predicted transmembrane spanning helices are underlined, and the Lag1 motif (amino acids 53–104) and the C-terminal acidicdomain (amino acids 187–203) are highlighted in gray. *The stop codon.

formation of human hepatoma cells in vitro. We also discuss reading frame encoding 230 amino acids. The predicted 27-
here the biological importance of this LAG1 homologue.
RESULT

Identification and Cloning of LASS2Using expressed sequence tag direct sequencing and bioinfor-matics analysis, we identified a liver cDNA clone, SP260, con-taining the 3� incomplete sequence of a previously unknownhuman homologue of the yeast LAG1. To distinguish it fromLAG1Hs-1, we called it LASS2. Based on the sequence of thiscDNA clone, we used rapid amplification of cDNA ends(RACE) PCR to clone the 5� end of LASS2 cDNA. The DNAsequence (Fig. 1) was a combined sequence of the clone fromliver cDNA library and that of the RACE product. We desig-nated the first ATG codon (bp 385–387) with flankingsequences consistent with Kozak consensus as the methionineinitiation codon. The full-length cDNA of LASS2 has an open

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kDa LASS2 protein contains four transmembrane helices, aLag1 motif, and a C-terminal acidic domain (Fig. 1).

By accessing the GenBank database for full-length cDNAs,we found that LAG1 homologues were transcribed in fish,mammals, insects, higher fungi, and plants. LASS2 had thehighest sequence identity to the Suberites domuncula, C. ele-gans, A. thaliana, and Halocynthia roretzi homologues (40%,36%, 36%, and 35% identity over 219, 211, 205, and 217 aminoacids, respectively; Fig. 2). Not only the LAG1 proteins amongdistantly related species but also some in the same speciesshowed very limited sequence identity. For example, LASS2showed only 26% identity to baker’s yeast Lag1p over 209amino acids, and 29% identity to the LAG1Hs-1 over 224amino acids (Fig. 2). However, most of them share a similarprofile of six or seven transmembrane helices and a basicdomain between the first two helices on the N terminus,except for human LASS2 and the sponge S. domunculaLAG1Sd, which have only four and five transmembrane

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helices, respectively. In addition, a consensus Lag1 motif and 10111–01001–01010–01000–10000–00010–01011–00011–01000–
a C-terminal acidic domain are present in most known LAG1homologues. The Lag1 motif is conserved in evolution, indi-cating an important function for LAG1 in a basic cellular func-tion in eukaryotes [2,3].
Tissue Expression Pattern of LASS2We examined the tissue expression pattern of LASS2 byhybridizing its cDNA probe to a multiple-tissue northern blot.We detected a transcript of ~ 2.4 kb in human kidney, liver,brain, heart, placenta, and lung (Fig. 3). The blot also showeda high level of expression in kidney and liver, and a low levelof expression in brain, heart, placenta, and lung. These resultsindicate LASS2 has an expression pattern different from thatof LAG1Hs-1, which is expressed mainly in brain, skeletalmuscle, and testis.

Chromosome Localization of LASS2We determined the chromosomal localization of LASS2 byradiation hybrid (RH) mapping. Data of LASS2 RH mappingobtained from the 93 RH clones in the GeneBridge 4 RH panelwere 00010–10001–01101–00110–00001–10110–01110–00010–

FIG. 2. Amino acid alignment of the LAG1 homologues of various organisms. Residues identical in � 50% of the compared sequences are on a black background,and similar residues are on a gray background (GenBank acc. nos.: LAG1Hs-1 and LASS2 (Homo sapiens, P27544, AF177338); LAG1Sd (S. domuncula, CAC03512);LAG1Ce-1 (C. elegans, AAD16893); LAG1At-2 (A. thaliana, AF198180); LAG1Hr (H. roretzi, BAA81907); and LAG1Sc (S. cerevisiae, P38703)).

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00000–010. We sent the mapping data to the WhiteheadInstitute RH Mapping Sever for analysis. The chromosomalassignment and placement data showed that LASS2 waslocated 7.04 cR from WI-497 and 4.40 cR from NIB288, andthat this gene was mapped to chromosome 1q11, in the ordercen–WI-497–LASS2–NIB288. GenBank database searches onNational Center for Biotechnology Information unfinishedhuman genome sequences showed that the LASS2 cDNAmatched two deposited genomic sequences, chromosome 1clone RP11-296P18 (GenBank acc. no. AC027437) and chro-mosome 5 clone RP1-253M23 (GenBank acc. no. AC020981),in 10 separate regions spanning about 4.2 kb. The exon–intronjunctions matched the GT-AG rule perfectly (Table 1). TheseRH mapping results indicated that LASS2 was mapped tochromosome 1q11, not chromosome 5.

Identification of LASS2-Interacting Protein through Yeast Two-Hybrid ScreeningScreening of the cDNA libraries using LASS2 as “bait”yielded 43 positive clones (23 from 4 � 106 fetal brain cDNAlibrary transformants and 20 from 1 � 106 liver cDNA library



transformants). Of the 43 clones, 41 passed the mating test for
interaction specificity. GenBank database searches showedthat 14 of the 41 clones encoded seven proteins of knownfunction. All were membrane-associated receptors or trans-porters. Among them, four were confirmed to physicallyinteract with LASS2 in the GST pull-down assay as describedbelow: the high-affinity asialoglycoprotein receptors type 1(AGPRH1; GenBank acc. no. M10058) and type 2 (AGPRH2;GenBank acc. no. U97179), the organic cation transporter-1(OCT1; GenBank acc. no. U77086), and the proteolipid sub-unit of vacuolar H+ATPase (VPL; GenBank acc. no. M62762).The 5� ends of the library cDNA inserts begin at positions –97bp (AGPRH1), –79 bp (AGPRH2), +917 bp and +973 bp (OCT1,two separated clones), and –112 bp (VPL) with respect to theinitiation codons.
AGPRH1, AGPRH2, VPL, and the C-terminal Half ofOCT1 Bind to LASS2 in a GST Pull-Down AssayTo confirm the physical interaction between LASS2 and theproteins identified as “prey” in the yeast two-hybrid screen-ing, we used a GST pull-down assay with biotinylated in vitrotranslated LASS2 and GST fusion proteins of AGPRH1,AGPRH2, VPL, and the C-terminal half of OCT1 (amino acids306–544). Confirming the interactions identified in the two-hybrid assay, we found that LASS2 bound to GST–AGPRH1,GST–AGPRH2, GST–VPL, and GST–OCT1 (amino acids306–504), but not to GST alone (Fig. 4). It has been reportedthat the E. coli S30 extract contains an endogenous biotiny-lated protein of 22.5 kDa (Technical Bulletin No. 219,Promega). In our pull-down assay, this endogenous protein

GENOMICS Vol. 77Copyright © 2001

TABLE 1: Exon–intron organization of the human LASS2a

Exon no./size (bp) 5� Splice donor Intron no./size (bp) 3� Splice acceptor

ex. 1 (107) CTTTGAGCT gtaagcatg... in. 1 (405) ...cttccccag GTACGTGGC

ex. 2 (118) CCCAAGCAG gtatgagcc... in. 2 (193) ...tgtgcccag GTGGAAGTA

ex. 3 (119) AGAAGCCAG gtgggggag... in. 3 (205) ...ctctgccag CTGGAGATT

ex. 4 (58) ATT GTG GAT gtgagtggg... in. 4 (105) ...tctctgcag AAA CCC TGG

I V D6 K7 P W

ex. 5 (51) CCC ATA CAG gtatgagtt... in. 5 (179) ...ctctcccag AGC ACT ATC

P I Q23 S24 T I

ex. 6 (93) AAG CGA AAG gtgggtgaa... in. 6 (189) ...tctctgcag GAT TTC AAG

K R K54 D55 F K

ex. 7 (129) CTG CTG GAG gtcaggctt... in. 7 (211) ...tctatgcag TCA GCC AAG

L L E97 S98 A K

ex. 8 (107) CCC TTC TG gtgagtagg... in. 8 (126) ...aatcctcag G ATC CTG

P F W133 I134 L

ex. 9 (154) ACT GGA AAG gtgaagccc... in. 9 (187) ...tttccacag CTG GTA GAA

T G K184 L185 V E

ex. 10 (1010)aExon/intron sizes and nucleotide sequence at the exon (uppercase) and intron (lowercase) boundaries are given.

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did not bind to the fusion proteins and GST, which may pro-vide additional evidence of the binding specificity.

Colony Formation Assay of Hepatocellular CarcinomaSMMC7721 Cells Transfected with LASS2 cDNATo determine the inhibitory effects of LASS2, we examinedthe colony-forming efficiency by transfecting LASS2 intohepatocellular carcinoma (HCC) SMMC7721 cells (Fig. 5). Thenumber of colonies formed by LASS2-transformedSMMC7721 cells was reduced to ~ 20% of that of the vectorcontrol (Fig. 5). The results were reproducible and indicatedthat the transfection of the pCMV-Script plasmid containingLASS2 into human HCC SMMC7721 cells was able to sup-press the growth of the cancer cells.

FIG. 3. Tissue distribu-tion of human LASS2mRNA. Data representnorthern blot analysisusing a human multiple-tissue northern blot con-taining 2 �g poly(A)RNA from differenthuman tissues in eachlane.

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DISCUSSION

We have cloned and characterized LASS2, a previouslyunknown human homologue of yeast LAG1 distinct from thehuman homologue LAG1Hs-1. The LASS2 transcript washighly expressed in liver and kidney. This expression patternof LASS2 is different from that of LAG1Hs-1, which isexpressed in brain, skeletal muscle, and testis but not in liverand kidney [3]. Given the differences in sequences andexpression patterns, LASS2 might be a distant relative ofLAG1Hs-1 with different biological functions.

The LASS2 protein lacks the N-terminal basic domainand two flanking transmembrane helices present in mostknown LAG1 homologues. The remaining four helices andother conserved domains indicate that LASS2 is a membraneprotein. This was partially confirmed by the yeast two-hybrid screening, which showed that LASS2 interacted withseveral membrane-associated receptors or transporters.Among them, four interacted with LASS2 in the GST pull-down assay, whereas others, including two other isoformsof the AGPRH2, failed to be expressed as GST fusion pro-teins in E. coli, and their physical interactions with LASS2remain to be confirmed.

It is not apparent at present what common denominatorconnects these membrane-associated proteins, but our resultsmay provide some indication of the biological function ofLASS2 in humans. Most of the membrane-associated LASS2-interacting proteins are responsible for the cellular transportof various substrates. Recent studies have shown thatlongevity can be modulated by various environmental cues,such as nutrition, heat, and desiccation [9]. Some receptors ortransporters are able to regulate the longevity of organisms.Examples of this include DAF-2 and DAF-12, two membersof the dauer formation pathway in the nematode C. elegans,which encode an insulin receptor homologue and a nuclearreceptor, respectively [9,10].

FIG. 4. AGPRH1, AGPRH2, VPL, and the C-terminal half of OCT1 specificallybind to LASS2 in vitro. GST fusion proteins bound to glutathione–Sepharose 4Bbeads were incubated with in vitro translated, biotinylated LASS2. LASS2 bindsspecifically to AGPRH1 (lane 2), AGPRH2 (lane 4), VPL (lane 5), and the C-ter-minal half of OCT1 (lane 3), but not to GST alone (lane 1). Lane 6, in vitro trans-lation product (control); LASS2 appears as a 27-kDa band. The 22.5-kDa bandin lane 6 represents an endogenous biotinylated protein in the E. coli S30 extract.

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Although there are many mechanisms of aging, thebroad physiological principles underlying aging in speciesranging from yeast to human may be similar [9].Identification of the structurally related and evolutionarilyconserved LAG1 homologues will help to demonstrate thesimilarities and differences in aging of various species.Furthermore, it will be necessary to manipulate LASS2 inmammalian systems to study its effects on longevity. In thispreliminary study, LASS2 was able to inhibit the colony for-mation of human hepatoma cells. This result provides a cluelinking this gene to the regulation of cell growth, as in can-cer. The molecular mechanism of the growth-suppressingeffect on HCC cells remains to be elucidated; these studiesare now in progress.

MATERIALS AND METHODS

Isolation of a full-length cDNA of LASS2. A cDNA fragment of 754 bp wasidentified from a human liver cDNA library (Life Technologies, Grand Island,NY) by expressed sequence tag direct sequencing and bioinformatics analysis.The fragment was previously called SP260. The incomplete coding sequenceof SP260, assembled by searching the public dbEST, encodes a polypeptidewith partial sequence identity to yeast LAG1; we called it LASS2. To isolate full-length cDNA of LASS2, primers for 5�-RACE, R1 and R2, were designed fromthe 3� untranslated region of this cDNA fragment. First-round 5�-RACE PCRused primers AP1 (adaptor primer 1, 5�-CCATCCTAATACGACTCAC-TATAGGGC-3�; Clontech, Palo Alto, CA) and R1 (5�-GGAAGGGGGAA-GAGGCCAGAGAAAG-3�; bp 1757–1733) with human liver Marathon-readycDNA (Clontech) and Advantage 2 polymerase mix (Clontech). The cyclingconditions consisted of one cycle of 94°C for 1 min; five cycles of 94°C for 30 sand 72°C for 4 min; five cycles of 94°C for 30 s and 70°C for 4 min; and twenty-seven cycles of 94°C for 20 s and 68°C for 4 min. Nested RACE PCR usedprimers AP2 (adapter primer 2, 5�-ACTCACTATAGGGCTCGAGCGGC-3�;

FIG. 5. Colony formation assay of transfected HCC SMMC7721 cells. Cellswere transfected with the indicated DNA and grown in the presence of G418for ~ 2 weeks. G418-resistant colonies were then examined with an invertedmicroscope and assigned scores. Data represent the average of three inde-pendent assays; error bars indicate the standard deviations.



reporters with only the LASS2 bait were chosen for further analysis. Plasmid
Clontech) and R2 (5�-TCCCCAGTACAGCCCCCACTTTTTG-3�; bp 1657–1633)and the same conditions as the first-round 5�-RACE PCR. The nested RACEamplification product was purified and cloned into the vector pT-Adv(Clontech) and sequenced using M13 reverse and forward primers. After 5�-RACE PCR, a full-length cDNA of LASS2, 1977 bp, was then constructed.
Sequence analysis. Protein sequences were analyzed using BLAST, PILEUP,and HELIXMEM. Online BLAST searches on GenBank database were madethrough the National Center for Biotechnology Information at the NationalInstitutes of Health (Bethesda, MD). Multiple sequence alignments were madeusing the PILEUP program of the UWGCG package. Prediction of trans-membrane helices used the HELIXMEM algorithm of PC/Gene software.

Northern blot analysis. A multiple-tissue northern blot (Clontech) washybridized with the LASS2 cDNA as a probe. This blot contains poly(A)-selected RNA from 12 different human tissues. The LASS2 cDNA probe waslabeled with [�-32P]dCTP by random priming (Amersham, Arlington Heights,IL). The RNA hybridization buffer contained 6� SSC, 5� Denhardt’s solu-tion, 50% formamide, 0.5% SDS, and 100 �g/ml denatured, sheared, salmonsperm DNA. Hybridization, membrane washing, and autoradiography weredone according to published methods [11].

Radiation hybrid mapping. Chromosomal mapping was accomplished byPCR screening of the GeneBridge 4 RH screening panel (Research Genetics,Huntsville, AL) [12] using primers A (5�-TCATTCTGCCAAAGCTGGAC-CAAGG-3�; bp 1484–1508) and R2 (5�-TCCCCAGTACAGCCCCCACTTTTTG-3�; bp 1657–1633). The cycling conditions used followed the manufacturer’sinstructions. A product of 174 bp was visualized on an ethidium bromide-stained agarose gel. Data were entered into the server of the WhiteheadInstitute/Massachusetts Institute of Technology Center for analysis with theGenome Research Radiation Hybrid Mapping Program (RHMAPPER;http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl).

Yeast two-hybrid screening. DupLEX-A (OriGene, Rockville, MD), a LexA-based yeast two-hybrid system, was used in library screening. To construct theLexA–LASS2 hybrid (pEG202-LASS2), the complete coding sequence of LASS2(bp 385–1141) was prepared by PCR and subsequently cloned into theEcoRI–XhoI cloning sites of the pEG202 (HIS3, ADH1-LexA) vector and verifiedby sequencing. The following primers were used for PCR amplification (EcoRIand XhoI sites are underlined): P57 (5�-CCGAATTCATGGCCGTCATTGTG-GATAAAC-3�) and P58 (5�-TTCTCGAGCGCAGGGAGCAGGGTAGTT-3�).

The “bait” plasmid pEG202-LASS2 was cotransformed into the yeast strainEGY48 (MATa, trp1, his3, ura3, and leu2::6LexAop-LEU2) together with thereporter gene plasmid pSH18-34 (URA3 and 8LexAop-GAL1-LacZ) or plasmidpJK101 (URA3 and GAL1-2LexAop-LacZ) to construct the selection strain orrepression assay strain, respectively. Yeast-competent cells were prepared witha lithium acetate method [13]. No autonomous transcriptional activation wasdetected in the selection strain (� 5 � 10–7 frequency on SG–His, –Ura, –Leuplates). In addition, the �-galactosidase activity in the repression assay strainwas decreased to a lower level (from deep blue to light blue on SG–His, –Ura,X-gal plates), which indicated the LexA–LASS2 hybrid was able to enter thenuclei and bind to the LexA binding sites (LexAop) on plasmid pJK101. Hence,the ‘bait’ was suitable for the detection of protein interaction in the two-hybridassay.

Two cDNA libraries in the plasmid pJG4-5 (TRP1 and GAL1-B42), 3� ofthe B42 activation domain, were derived from human fetal brain and liver,respectively (OriGene). For the yeast two-hybrid screen, the selection strainwas transformed with the cDNA libraries. The resulting 4 � 106 (for fetalbrain cDNA library) and 1 � 106 (for liver cDNA library) transformants wereplated on selective galactose plates to screen for activation of leucine (LEU2)reporter gene. Colonies that appeared before 10 d were tested for the galac-tose-dependent expression of another reporter gene, �-galactosidase (LacZ),using replica on X-gal plates. For the positive (blue) colonies, the “bait” plas-mid pEG202-LASS2 and reporter plasmid pSH18-34 were eliminated bygrowth in non-selective medium. The resulting strains that contained only thelibrary plasmid were mated with strain RSF206 (MATa, trp1∆::hisG, his3∆200,ura3-52, lys2∆201, and leu2-3), which has an opposite mating type and con-tains the reporter plasmid pSH18-34 together with one of the following plas-mids: pRHM1, pEG202-Max, pBait, or pEG202-LASS2. The first three, pro-vided by OriGene, are pEG202-based plasmids containing unrelated baits.The clones that showed specific galactose-dependent activation of the two

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DNA was isolated from yeast by glass bead lysis, water boiling, and ethanolprecipitation [14], and was transformed into the E. coli strain DH5� by theCaCl2 method. We randomly chose colonies for each cDNA clone, then didplasmid minipreps and analyzed the size of cDNA inserts after PCR ampli-fication. Plasmids containing each size cDNA insert from each clone weresequenced. The primers used for PCR amplification and sequencing wereP49 (5�–CTGAGTGGAGATGCCTCC-3�) and P50 (5�–GCCGACAAC-CTTGATTG-3�).

GST pull-down assay. The cDNAs containing the complete coding sequencesof AGPRH1 (bp –2 to +1106), AGPRH2 (bp –11 to +1186), and VPL (bp –6 to+912), as well as the incomplete coding sequence of OCT1 (bp +918 to +1635)were subcloned into the pGEX-5X-1 vector (Pharmacia Biotech, Uppsala,Sweden). The GST fusion proteins were expressed in 0.1 mM isopropyl-1-thio-�-D-galactopyranoside (IPTG)-induced E. coli strain BL21 and were purified byglutathione–Sepharose 4B beads (Pharmacia Biotech). LASS2 in plasmidpT7XLASS2 (a gift from Yi Gong, Research Center of Biotechnology, ChineseAcademy of Sciences, Shanghai, China) was translated in vitro with the E. coliT7 S30 extract system for circular DNA (Promega, Madison, WI) and TranscendBiotin-Lysyl-tRNA (Promega). Equivalent amounts of GST–AGPRH1,GST–AGPRH2, GST–VPL, GST–OCT1 (amino acids 306–544), or GST alonewere immobilized on glutathione–Sepharose 4B beads and incubated for 2 hat 4°C with gentle rocking with 5 �l biotinylated in vitro-translated LASS2 in50 �l NETN buffer (100 mM NaCl, 1 mM EDTA, 20 mM Tris Cl, pH 7.5, 0.5%Nonidet P-40, 1 mM DTT, and 1 mM phenylmethylsulfonyl fluoride). Beadswere washed three times in 100 �l buffer H (20 mM HEPES, pH 7.7, 50 mMKCl, 20% glycerol, 0.1% Nonidet P-40, and 0.007% �-mercaptoethanol) andboiled in 20 �l Laemmli buffer, and proteins in the supernatant were separatedby electrophoresis through SDS–12% polyacrylamide gels. The separated pro-teins were transferred to PVDF membrane and biotinylated proteins weredetected with the Transcend chemiluminesent translation detection system(Promega).

Colony-formation assay. LASS2 cDNA containing the complete codingsequence (bp 1–1223) was subcloned into the expression vector pCMV-Script(neo+; Stratagene, La Jolla, CA). The plasmid DNA was extracted and purifiedwith the Qiagen plasmid purification system. HCC SMMC7721 cells (a giftfrom Shanghai Second Military Medical University) grown in a 96-well platewere transfected with pCMV-Script/LASS2 plasmid DNA, pCMV-Script/p53plasmid DNA, or pCMV-Script void vector DNA by means of LipofectAMINEin conditions recommended by the manufacturer (Life Technologies). PlasmidDNA (100 ng) was dissolved in 6 �l distilled water and mixed with 0.74 �lLipofectAMINE reagent in 9.3 �l serum-free media, and incubated at roomtemperature for 10 min. Then, 150 �l serum-free media was added, and theDNA–liposome solution was equally applied to a monolayer of 1 � 103

SMMC7721 cells in three wells of a 96-well plate. Each well was supplementedwith 50 �l serum-free medium after 2 h of incubation. The next day the DNAsolution was removed and the cells were supplied with fresh, completemedium. After 24 h, the transfected cells were selected in media containing 800�g/ml G418. Cells were incubation for 14 d and colonies were examined withan inverted microscope and assigned scores [15,16].

ACKNOWLEDGMENTSThis work was supported by the Chinese National Human Genome Center atShanghai Grant (CNCZ-99Z-01) to K.-K.H. and K.T.G., National EducationMinistry Key Laboratory Visiting Scholar Grant (99-105) to K.T.G., and ChinaState Key Basic Research Program Grant (G1998051209) to D.-F.W.

RECEIVED FOR PUBLICATION DECEMBER 5, 2000; ACCEPTED JUNE 28, 2001.

Note added in proof. While this paper was being prepared, we found cDNAsequences in the GenBank data library with very high levels of sequenceidentity to LASS2 cDNA: BC001357, XM_002024, AK001105, and AF189062.

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Copyrig64

Sequence data from this article have been deposited with the EMBL/GenBank Data Libra

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ries under accession number AF177338.

Documents

Cloning, Mapping, and Characterization of a Human Homologue of the Yeast Longevity Assurance Gene LAG1