BRIEF REPORTS TN

Cloning and Characterization of a Novel Human cDNA That Has DNA Similarity to the Conserved Region of the Collagenase Gene Family

TN

Nancy Smyth Templeton,’ Lisa A. Rodgers, Anna T. Levy, Kai-Li Ting,* Henry C. Krutzsch, Lance A. Liotta, and William G. Stetler-Stevenson Laboratory of Pathology National Cancer Institute, Maryland 20892 Received

February

and *Laboratory of Mathematical Biology, National Institutes of Health, Bethesda,

25, 1991;

revised

August

FIG. 1. Northern blot analysis of total cellular RNA from colon tumor tissue (T) and adjacent normal colonic mucosa (N). Samples shown were from a patient diagnosed with Duke stage C2 adenocarcinema of the colon. 5 pg of each sample were size fractionated through a 1% w/v agarose gel containing 2.0 M formaldehyde and transferred to GeneScreen Plus membranes. The membrane was hybridized with [a-32P]dCTP-labeled cDNA probes at 42°C and washed at 65°C for 30 min in 0.2X SSC. Right: 3.1-kb message for the 72-kDa type IV collagenase. Also shown is the P-actin message (0.9 kb), which was used to normalize sample loading. Left: the same samples hybridized with [cu-32P]dCTP-labeled pM5. Normalization of the signal intensities following laser densitometric scanning showed that the normal tissue contained a 1.5-fold increase in pM5 signal compared with the colon tumor tissue. Therefore, there is no significant difference between normal and tumor colon tissues in expression of pM5 mRNA.

5, 1991

A novel human cDNA was isolated from an A2058 melanoma cDNA library using the pCD-X Okayama-Berg vector. The library was screened with oligonucleotide probes that have homology to the metal-binding domain of human fibroblast coihgenase (Templeton et al., 1990), a matrixdegrading metalloproteinase. The metal-binding domain is highly conserved among all members of the collagenase family. The probes used for screening include: WSS-15, GAGTCCAAGAGAATGGCCGAGTTCACTAGCCGC; WSS-16, GAGTCCAAGAGAATGGCCGATTCCACTAGCCGC; and WSS-20, GAGTCCAAGAGAATGGCCGAGTCCATGAGCCGC. Three probes were used because the DNA sequences for the metalloproteinase metal-binding domain are degenerate, and these probes are complementary to the metal-binding domain. Two positive clones were isolated from the library screen. One clone contains the interstitial collagenase (Templeton as pM5. A restricet al., 1990), and the other was designated tion map of pM5 was generated, and overlapping Ml3 clones were made. The single-stranded Ml3 clones and double-stranded pM5 were sequenced on the Genesis 2000 automated sequencer (DuPont) using the fluorescent chainterminating dideoxynucleotide method (Prober et al., 1987; Sanger et al., 1977). Both strands have been sequenced with about 75 bases overlapping on each sequence run, and all restriction sites have been sequenced across. Each strand was sequenced at least twice, and all bases were checked manually. The nucleotide sequence described here has been submitted to the GenBank Data Libraries under Accession Number X57398. The entire pM5 sequence consists of 4086 bp. The open reading frame extends from nucleotides 1 to 3570, and this could encode a protein of 1190 amino acids. The first methionine is found at amino acid 47. The poly(A) signal is found 4042 to 4047. Nucleotides 3493 to 3500 are at nucleotides 100% homologous to the complement of nucleotides 14 to 21 of WSS-15 and WSS-20. Nucleotides 1249 to 1257 of pM5 are 100% homologous to the complement of nucleotides

22 to 30 of WSS-20.

Northern

blot

analysis,

pM5 DNA as a probe, shows one band approximately 4 kb in length (Fig. 1). Both human normal and malignant colorectal tissues show the same level of expression of the pM5 message. GenBank (Release 67.0, 3/91; Burks et al., 1991) and Swiss-Prot (Release 17.0, Z/91; Bairoch and Boeckmann, 1991) were compared with the pM5 DNA and protein sequences, respectively. No significant homologies were found by using the UWGCG package Version 7.0 Wordsearch command (Wilbur and Lipman, 1983). The highest score found was 65, indicating only extremely dissimilar sequences. Among the most similar sequences were several collagens that were detected only because of their composition; their similarities are at this extremely low level. The longest identical sequence found was only five residues. Internal similarities on the pM5 sequence were also investigated with the SEQHP program (Kanehisa and Goad, 1982; Goad and Kanehisa, 1982); the longest identical segments again were only five residues long. REFERENCES

using

’ To whom correspondence should be addressed at Molecular Hematology Branch, National Heart, Lung and Blood Institute, NIH, Bldg. 10, Rm. 7D18, Bethesda, MD 20892.

1.

BAIROCH, A., AND BOECKMANN, tein sequence data bank. Nucleic 2249.

2.

BURKS, C., CASSIDY, M., CINKOSKY, M. J., CUMELLA, K. E., GILNA, P., HAYDEN, J. E.-H., KEEN, G. M., KELLEY, T. A., KELLY, M., KRISTOFFERSON, D., AND RYALS, J. (1991). GenBank. Nucleic Acids Res. lS(Supp1.): 2221-2225. GOAD, W. B., AND KANEHISA, M. I. (1982). Pattern recognition in nucleic acid sequences. I. A general method for finding

3.

175

B. (1991). B Swiss-Prot, Acids Res. lS(Supp1.):

GENOMICS

pro2247-

12,175-176 (1992) 0888-7543/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

176

BRIEF

local homologies and symmetries. Nucleic

Acids

Res. 10: 247-

263. 4.

KANEHISA,

M.

I., AND

GOAD,

W.

B. (1982).

Pattern

recogni-

tion in nucleic acid sequences. II. An efficient method for finding locally stable secondary structures. Nucleic Acids Res. 10: 265-278. 5.

PROBER, J. M., TRAINOR, G. L., DAM, R. J., HOBBS, F. W., ROBERTSON, C. W., ZAGURSKY, R. J., COCUZZA, A. J., JENSEN, M. A., AND BAUMEISTER, K. A. (1987). A system for

rapid DNA sequencing with fluorescent chain-terminating clideoxynucleotides. Science 238: 336-341. 6.

SANGER, sequencing

Acad.

F., NICKLEN, S., AND with chain-terminating

Sci. USA

COULSON, A. R. (1977). inhibitors. Proc.

DNA

N&l.

74: 5463-5467.

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TEMPLETON, N. S., BROWN, P. D., LEVY, A. T., MARGULIES, I. M. K., LIOVA, L. A., AND STETLER-STEVENSON, W. G. (1990). Cloning and characterization of human tumor inter-

8.

WILBUR,

stitial collagenase. Cancer Res. 50: 5431-5437. W.

J., AND

LIPMAN,

D. J. (1983).

Rapid

similarity

searches of nucleic acid and protein data banks. Proc. Natl. Acad.

Sci. USA

80: 726-730.

Highly Polymorphic Sequence in lntron MAOB Gene

(CT), Repeat II of the Human

C. Konradi,* L. Ozelius,**t and X. 0. Breakefield**$ ‘Departments of Neurobiology Genera/ Hospital, Charlestown, Department and #Neuroscience Boston, Massachusetts 02 115 ReceivedJune

24, 1991;revised

and Neurology, Massachusetts Massachusetts 02129; and tGenetics Program, Harvard Medical School,

September

6, 1991

Monoamine oxidase (MAO; monoamine: 0, oxidoreductase, EC 1.4.3.4) is the primary enzyme involved in the degradation of aminergic neurotransmitters like dopamine, noradrenaline, and serotonin, and the neurotoxin precursor, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Heikkila et al., 1985). Two isozymes, MAO-A and MAO-B, differ in their pharmacologic and biochemical properties (for review see Weyler et al., 1990). Genes for MAO-A and MAO-B are located near each other in the ~11.3 region of the human X chromosome (for review see Hsu et al., 1989). Over 50-fold variations in MAO-A and MAO-B activities have been described in control humans, as measured in cultured skin fibroblasts and platelets, respectively, and activity levels appear to be genetically determined (Hsu et al., 1989). Since variations in MAO activity can influence neurophysiology, it seems likely that individuals with different levels of activity may have differential susceptibilities to certain pathogenic processes. Biochemical measurements of MAO-B activity in peripheral tissues have not resolved the possible role of MAO-B in human diseases, presumably because a number of factors, including drugs, hormonal state, age, and diet influence levels of MAO-B activity in vivo (for review see Demisch et al., 1983). Molecular biological methods can be used to follow the inheritance of the MAOB gene in families and to evaluate rapidly the associaGENOMICS 12,176-177 (1992) 0888-7543/92 $3.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form

Inc. reserved.

REPORTS

tion of different alleles with disease states. Here we describe a highly polymorphic (GT), repeat (Weber, 1990) within the second intron of the human MAOB gene and define primers and conditions for amplification of it using the polymerase chain reaction (PCR; Saiki et al., 1988). A human genomic DNA clone, MAOB-5, was isolated from an EMBL 3A phage library, generated from partially Sau3A-digested genomic DNA cloned into a BamHI site (Kwiatkowski et al., 1988) by screening with a partial cDNA clone, KSB2 (kindly provided by Dr. Paul Hsu, West Roxbury Veterans’ Administration, MA), corresponding to nucleotides lo-600 in the 5’ end of the published human MAO-B cDNA (Bach et al., 1988). This 30-kb genomic clone was found to contain a (GT), repeat by screening with a poly(dC-dA) . (dG-dT) oligonucleotide probe (Pharmacia, Piscataway, NJ). Following digestion of clone MAOB-5 with EcoRI and PstI and gel electrophoresis, a 1.8-kb band that hybridized to both the partial MAOB cDNA clone and the (GT), oligonucleotide probes was identified. Southern hybridization was carried out as described (Hotamisligil and Breakefield, 1991). This band was subcloned into a Bluescript SK vector (Stratagene, La Jolla, CA). Sequencing of the 1.8-kb human genomic clone 2.2 revealed a (GT),, repeat located 264 bp from the 3’ end of exon II (Grimsby et al., 1991). Double-stranded sequencing was performed by the alkaline-denaturation method (Toneguzzo et al., 1988) followed by the chain termination reaction (Sanger et al., 1977) using [35S]dATP and modified T7 DNA polymerase (United States Biochemical, Cleveland, OH). No other MAO-B exon sequence was present in clone 2.2. Primers flanking the (GT), repeat (DNA synthesizer, Cyclone, Biosearch, San Raffael, CA) had the following sequence: 5’ GAA GCA TCG AAG TTA GGA GT 3’ (CA strand) and 5’ ATT TGG CCT CAT AGA GTT AG 3’ (GT strand). PCR reactions contained 0.2 mM dATP, dCTP, dTTP, 2.5 pM dTTP, 0.08 ~1 [32P]dTTP (3000 mCi/ mmol),4 ng of each primer, 10 ng genomic DNA, and 0.05 ~1 Tuq polymerase (Perkin-Elmer Cetus) in a total volume of 10 ~1. Samples were heated to 94°C for 1.5 min, followed by 25 cycles each for 1 min at 94”C, 1 min at 55°C and 1 min at 72”C, and a final extension for 7 min at 72°C. Seven different MAOB alleles were detected by acrylamide gel electrophoresis of PCR fragments generated from 48 randomly chosen, nonrelated controls (17 males, 31 females; total 79 X chromosomes). The sizes of the fragments ranged from 172 to 184 bp. The frequency of alleles in this control population is shown in Table 1. The expected heterozygosity for

TABLE of MAOB

Frequency

alleles

1 in control

Allele

Frequency

172 174 176 178 180 182 184

0.025 0.076 0.127 0.139 0.228 0.241 0.165

population

Cloning and characterization of a novel human cDNA that has DNA similarity to the conserved region of the collagenase gene family.

BRIEF REPORTS TN Cloning and Characterization of a Novel Human cDNA That Has DNA Similarity to the Conserved Region of the Collagenase Gene Family T...
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