GENOMICS

l&441-448

(1991)

Chromosomal JONATHAN

Localization F. TAIT,*‘t”

of the Human Annexin

III (ANX3)

Gene

D. ALAN FRANKENBERRY,* CAROL H. MIAO,$ ANN M. KILLARY,~ A. ADLER,t AND CHRISTINE M. DlsTECHEt

DAVID

Departments of *Laboratory Medicine, tfathology, and $Biochemistry, University of Washington, Seattle, Washington 98195; and §Division of Laboratory Medicine, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030 Received

August

10, 1990;

revised

February

4, 1991

tain (Flaherty et al., 1990). Several other functions have been proposed, including regulation of inflammation, regulation of exocytosis, membrane-cytoskeletal linkage, mediation of intracellular calcium signals, and transmembrane calcium transport (Crompton et al., 1988; Burgoyne and Geisow, 1989; Burns et al., 1989). Most recently, annexin III has been identified as inositol 1,2-cyclic phosphate 2-phosphohydrolase (EC 3.1.4.36), an enzyme of inositolphosphate metabolism (Ross et al., 1990). This raises the possibility that this protein and perhaps other annexins may be important in intracellular signaling (Ross et al., 1990). The annexin III protein has been previously isolated as human inositol 1,2-cyclic phosphate 2phosphohydrolase (Ross and Majerus, 1986), human placental anticoagulant protein III (PAP-III)3 (Tait et al., 1988), rat lipocortin III (Pepinsky et al., 1988), rat calcimedin 35-a (Kaetzel et al., 1989), and an abundant cytoplasmic protein from human neutrophils (Ernst et al., 1990). The human cDNA sequence (Pepinsky et al., 1988), which agrees with protein sequence data (Tait et al., 1988), predicts a protein of 322 amino acids with approximately 50% amino acid identity with other annexins. We have undertaken chromosomal mapping of the gene encoding annexin III (ANX3) as part of a study of its structure and function. Further insight into the physiological functions of the annexin protein family could come from linkage with human genetic disease. We have localized the ANX3 gene to human chromosome 4 by using PCR to amplify an intron-containing region of the gene from a panel of somatic cell hybrids (Iggo et al., 1989). In situ hybridization demonstrates that the ANX3 gene maps to the long arm of chromosome 4 at band q21 (q13-q22).

The annexins or lipocortins are a new family of calciumdependent phospholipid-binding proteins. Annexin III has been previously identified as inositol 1,2-cyclic phosphate 2-phosphohydrolase (EC 3.1.4.36), an enzyme of inositol phosphate metabolism, and also as placental anticoagulant protein III, lipocortin III, calcimedin 36-a, and an abundant neutrophil cytoplasmic protein. In this study, the gene (ANXS) encoding annexin III was localized to human chromosome 4 at band q21 (q13-q22) by (1) polymerase chain reaction analysis of a human-rodent hybrid cell panel, confirmed by genomic Southern blot analysis of the same panel with a cDNA probe and (2) in situ hybridization with a cDNA probe. 0 lS91 Academic Press, Inc.

INTRODUCIION

The annexins’ have recently been identified as a new family of calcium-dependent phospholipid-binding proteins (reviewed by Klee, 1988; Crompton et al., 1988; Burgoyne and Geisow, 1989). This family contains at least 10 distinct members (Haigler et al., 1989; Hauptmann et al., 1989; Johnston et al., 1990), each of which contains 4 or 8 copies of an 80-amino-acid repeating unit first identified in lipocortin/annexin I (Wallner et al., 1986). We and others have isolated several members of this protein family from placenta on the basis of their anticoagulant activity, which is due to phospholipid binding (Reutelingsperger et al., 1985, 1988, Funakoshi et al., 1987a,b; Iwasaki et al., 1987; Tait et al., 1988,1989; Maurer-Fogy et al., 1988; Hauptmann et al., 1989). The physiological role of these proteins as anticoagulants has remained uncerSequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession No. M63310. ’ To whom correspondence should be addressed. * “Annexin” followed by a Roman numeral has been proposed as a name for proteins of this family; cross references to other names for these proteins have been summarized (Crumpton and Dedman, 1990). In this system, annexin III corresponds to PAP-III and annexin V corresponds to PAP-I.

3 Abbreviations used: PAP, placental anticoagulant PCR, polymerase chain reaction; SDS, sodium dodecyl SSC, 15 n&f sodium citrate, pH 7.0, 150 m&f NaCl. 441

protein; sulfate;

OSSS-7543/91

Copyright All

rights

0

1991 of reproduction

by

Academic in any

form

$3.00

Press, Inc. reserved.

442

TAIT

MATERIALS

AND

METHODS

Oligonucleotides Oligonucleotides were synthesized on an Applied Biosystems Model 381A DNA synthesizer using phosphoramidite chemistry and reagents supplied by the manufacturer. Stepwise yield was usually >99.0% and oligonucleotides were used without further purification. Oligonucleotides were designed to contain 1921 bases at the 3’ end matching the cDNA sequence and an EcoRI or Hind111 restriction site at the 5’ end. For annexin III, the oligonucleotides used as PCR primers were 5’-TTG AAT TCG AAA GTC TGA AAG TGG ATG AGC-3’ (bases 542-563 of the cDNA sequence (Pepinsky et al., 1988)) and 5’-GGA AGC TTG CTG TCC ACA ATG TCC TTT TG-3’ (reverse complement of bases 707-727). For annexin V, the oligonucleotides used were 5’-TTG AAT TCG CCT ACC TTG CAG AGA CCC T-3’ (bases 757-776 of the cDNA sequence (Funakoshi et al., 198713)) and 5’CTA AGC TTA GTC ATC TTC TCC ACA GAG C-3’ (inverse complement of bases 954-975). DNA Probes The cDNA for annexin III (PAP-III) was the plasmid pPAP-III-28A, which contains a 1268-bp insert in the EcoRI site of pUC18. This cDNA contains the complete 969-bp protein-coding region as well as 36 bp of 5’ untranslated sequence and 263 bp of 3’ untranslated sequence. This clone was obtained by standard methods of immunoscreening (Sambrook et al., 1989) of a Xgtll human placental cDNA library (Clontech, Palo Alto, CA) with affinity-purified polyclonal rabbit antiserum against PAP-III purified as described (Tait et al., 1988). The longest clone obtained, PAP-III-28A, was completely sequenced; the translated amino acid sequence agreed with the partial protein sequence data previously reported (Tait et al., 1988) for PAP-III. The sequence of this clone is identical to bases 11 to 1278 of the human lipocortin III cDNA, with the exception of a T instead of the C reported at position 1057 in lipocortin III (Pepinsky et al., 1988). The cDNA for annexin V (PAP-I) was the plasmid pPAP-I-1.6, which contains a 1459-bp insert in the EcoRI site of pUC18 (Funakoshi et al., 1987b). This cDNA contains the complete 960-bp protein-coding region as well as 12 bp of 5’untranslated sequence and 487 bp of 3’ untranslated sequence. For Southern blot analysis, the probes used were cDNA inserts released by EcoRI digestion and purified by agarose gel electrophoresis and electroelution. For in situ hybridization, whole plasmids were used. There is 52% sequence identity between the annexin III and annexin V cDNA probes.

ET

AL.

Cell Lines and DNA Purified genomic DNA samples from a panel of 25 human-hamster hybrid cell lines were obtained from the BIOS Corp. (New Haven, CT). The cell lines were originally derived in the laboratory of Dr. John Wasmuth (University of California, Irvine) by fusing a Chinese hamster ovary cell line with peripheral blood leukocytes from human donors (Dana and Wasmuth, 1982; Carlock et al., 1986) and were characterized by karyotype analysis. The chromosome content of the current passage of each cell line at BIOS Corp. was determined by blinded analysis of 20 metaphases by Giemsa banding (Table 1). Cell line HA(4)A (formerly referred to as HA(30)) is a monochromosomal microcell hybrid in the A9 mouse fibroblast cell background. It contains a single, intact copy of human chromosome 4 as the only human genetic material in the hybrid as determined by sequential G-banding and G-11 staining analysis (DeLange et aZ., 1990). The presence of chromosome 4 in the hybrid was verified by PCR analysis (see Results). Human genomic DNA was prepared from peripheral blood samples of Caucasian individuals or families. Mouse genomic DNA was from a male mouse, strain C57BL/6. PCR, Electrophmesis, and Southern Blotting PCR was performed as described (Mullis and Faloona, 1987; Saiki et al., 1988). Reactions were prepared in sterile 0.6-ml microcentrifuge tubes with pipets used only for that purpose. Each 50-~1 reaction contained 100-500 ng genomic DNA, 1 U Taq polymerase (Perkin-Elmer, Norwalk, CT), 200 PM each dNTP (Pharmacia, Piscataway, NJ), 10 pmol each primer, 1.5 mM MgCl,, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.1 g/liter gelatin. Each reaction was overlaid with 40 ~1 mineral oil and subjected to amplification in a DNA thermal cycler (Perkin-Elmer) with a thermal profile optimized for each primer combination. All amplifications were performed in the instrument’s step-cycle mode and included an initial 6-min denaturation step at 94°C and a final IO-min extension step at 72°C. For annexin III, the thermal profile consisted of 30 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 2 min; for annexin V, the thermal profile was 30 cycles of 94°C for 1 min, 55°C for 0.5 min, 72°C for 3 min. Agarose gel electrophoresis was performedwith horizontal gels (11 X 14 X 0.7 cm) in 89 mM Tris base, 89 mM boric acid, 2 mM EDTA, pH 8.3, with subsequent visualization by ethidium bromide staining. Molecular weight markers consisted of X DNA (HindIII digest) plus 4X174 DNA (Hue111 digest). Restriction enzyme digestions were performed at 37°C for 2 h in buffers recommended by the supplier. Southern blots

CHROMOSOMAL

MAPPING

4.4-

2.0IA-

FIG. 1. PCR analysis of human-hamster hybrid panel. Genomic DNA (0.25 pg) from human, hamster, or the indicated numbered hybrid cell line was subjected to amplification with annexin III-specific primers as described under Materials and Methods. PCR product (15 ~1) was electrophoresed at 70 V for 4 h in a 1.4% agarose gel; gels were then stained with ethidium bromide. “Neg” is the negative control (PCR reaction without added DNA).

were prepared on a nylon membrane (GeneScreen Plus, NEN-DuPont, Boston, MA) by the alkalinetransfer method (Reed and Mann, 1985). Probes were labeled to a specific activity of > 1 X 10’ dpm/pg by the random-priming method. Blots were hybridized for 18 h at 65°C with 3 X lo6 dpm/ml of probe in a solution consisting of 0.1 M Tris-HCl, 0.1 M Tris base, pH 8.0, 0.9 M NaCl, 5 mMEDTA, 1% SDS, 0.5% nonfat dried milk, 5% dextran sulfate. The final wash step following hybridization was performed at 60°C in 0.1X SSC/l% SDS. In Situ Hybridization

OF

ANNEXIN

III

GENE

443

cloned cDNA template was found, providing presumptive evidence for amplification of an intron-containing region of the gene. PCR conditions were then optimized to yield a single major band from human genomic DNA templates that was also distinct from any bands observed with hamster DNA templates. For annexin III (Fig. l), a single major band of 2.0 kb was amplified from human genomic DNA and from cell lines 803 and 1006; hamster DNA and the remaining hybrid cell lines did not show any major bands. The identity of the presumptive gene product was verified by high-stringency probing of Southern blots of PCR products with the annexin III cDNA (Fig. 2). The 2.0-kb PCR product from human DNA and cell lines 803 and 1006 hybridized with the annexin III cDNA, whereas no hybridization occurred at 2.0 kb for PCR products from hamster DNA or randomly selected cell lines that lacked the 2.0-kb band on ethidium-stained gels. The 2.0-kb PCR product has also been unambiguously identified as the product of the annexin III gene by DNA sequencing of the cloned PCR product (L. Xu and J. F. Tait, unpublished data). A second faint band is visible at lower molecular weight in Fig. 2. This may represent the product of an unknown gene or pseudogene on a different chromosome that is similar to annexin III. However, it is unlikely to represent the product of another known annexin gene, because the annexin V cDNA did not cross-hybridize to the annexin III cDNA (Fig. 2). Annexin III is as closely related to annexin V as to any other known annexin (Hauptmann et al., 1989). These results indicate that the ANX3 gene is on

The whole plasmid probe pPAP-III-28A described above was labeled by nick translation with 3H-labeled nucleotides to a specific activity of 1 X lo7 cpm/pg. In situ hybridization to metaphase chromosomes from lymphocytes of a normal human male donor was carried out with the probe at a concentration of 0.03 ng/pl of hybridization mixture as described by Marth et al. (1986). The slides were exposed for 43 days and the chromosomes were identified by Q-banding. RESULTS Analysis of Somatic Cell Hybrid Panel We first sought PCR primers that would allow amplification of a distinctive human product in the presence of the homologous hamster gene. Although the structure of the annexin III gene is unknown, primer pairs could be designed from the known cDNA sequence. A primer pair that yielded an amplified product from human genomic DNA templates that was larger than the corresponding product from the

FIG. 2. Southern blot analysis of PCR products. PCR product (2 ~1) from annexin III amplifications was electrophoresed (1.1% agarose, 50 V for 7.5 h), blotted, and hybridized with the annexin III cDNA probe as described under Materials and Methods. The final wash was at 60°C in 0.1X SSC/l% SDS. Lanes labeled “AnnIII” and “AnnV” contained 380 pg of the corresponding cDNA as positive and negative controls for hybridization stringency.

Note. The p15.1-~15.2;

III

+

324

+

423

+

+

+

734

+

+ + +

d

750

+

+ +

15%

+

+ +

803

presence or absence of the annexin dq, multiple deletions in 5q.

20 12 24 0 80 24 8 12 20 20 12 24 24 36 16 20 16 24 28 20 28 16 12 24 12

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y Marker

Annexin

% Discordant

Human chromesome

Chromosome

III

+

+

+ +

+

60%

867

+

+

940

of Hybrid

gene in the hybrid

+

45%

15%

+ +

15%

860

Content

+

25%

40%

+

+

+

+

507

cell lines

+

dq

212

Panel

1

+

+ + +

+ + 45%

d + +

756

was determined

+ +

+

+

+ 45%

+

683

Somatic 983

by PCR

+ +

+

811

analysis

862

line

Discordance

cell hybrid

and Percentage

TABLE

937

as shown

909

+

+

854

in Fig.

-

for Annexin

967

1. Abbreviations:

+

+

5%

+

d +

904

III Gene

968

+

+

1049

d, chromosome

+

+

+

+

+

+ +

55% +

1006

5 deleted

60%

10% +

45%

+

+

1079

at

+ +

+

d

+

1099

2 F

3

3

CHROMOSOMAL

MAPPING

OF

ANNEXIN

III

445

GENE

In Situ Hybridization

Annexin V

Annexin III

FIG. 3. PCR analysis of human-mouse hybrid cell line HA(4)A. The cell line contains chromosome 4 as its only human chromosome. (A) Ethidium-stained gel of PCR products obtained with primers specific for annexin V (left) or annexin III (right). PCR and electrophoresis were performed as described in the legend to Fig. 1. (B) Southern blot analysis of annexin III PCR products.

human chromosome 4 (Table 1). There is 0% discordance for chromosome 4 and at least 8% discordance for all other chromosomes. The localization of annexin III to chromosome 4 was verified by PCR analysis of a mouse-human hybrid cell line (HA(4)A) containing chromosome 4 as its only human chromosome. The presence of chromosome 4 in this cell line was verified by PCR analysis with annexin V-specific primers, which gave a 1.5kb product from human DNA and the HA(4)A hybrid (Fig. 3A, left); the gene for annexin V is known to be on chromosome 4 (Modi et al., 1989). With annexin III-specific primers, the same human-specific 2.0-kb band previously observed in the hamster-human hybrids was also seen in the mouse-human hybrid cell line (Fig. 3A, right), confirming that annexin III maps to chromosome 4. Southern blot analysis showed that the 2.0-kb PCR product from the HA(4)A cell line also hybridized with the annexin III cDNA (Fig. 3B). Although the PCR analysis provided an unambiguous localization to chromosome 4, supplementary data were also obtained by Southern blot analysis of HindIII-digested genomic DNA from the same 25 human-hamster hybrid cell lines (Fig. 4). Human-specific bands of 6.0, 5.6, 5.0, and 4.2 kb were present in cell lines 803 and 1006 and were absent in all other cell lines, which is the same pattern observed in the PCR analysis. The other hybrid cell lines not shown in Fig. 4 were also negative for the human-specific bands. Human bands at 8.0, 3.8, 3.6, 3.1, 2.1, 1.1, 0.9, and 0.6 kb were not scored because they were either too faint or too close in size to hamster-specific bands.

In situ hybridization of a cDNA probe for annexin III provided an independent mapping assignment of the gene and allowed its sublocation on chromosome 4. A total of 98 cells were examined for annexin III. Of 138 sites of hybridization, 37 (27% of the sites) were located in the proximal region of the long arm of chromosome 4 between band 4q13 and 4q22 (Fig. 5a). The largest number of grains (28 grains) were at band 4q21 (Fig. 5b). Except for a small peak of hybridization (3% of grains) that was seen at the distal end of chromosome 1, there were no peaks of hybridization on other chromosomes. These results also indicate that the annexin III cDNA does not cross-hybridize significantly to other known or unknown members of the annexin family. Relationship to ANX2Ll

Gene

The annexin II cDNA detects four independently segregating loci, one of which (ANXBLl) is on chromosome 4 in region q21-q31.1 (Huebner et al., 1988). To determine whether this locus might be the same as the ANX3 gene, we compared their genomic restriction maps as determined by Southern blot analysis with cDNA probes. For HindIII, the 3.6-kb band detected by annexin III (see above) is very similar to the 3.4-kb Hind111 band detected by annexin II and linked to chromosome 4. For EcoRI, the annexin III cDNAdetectsbandsof8.7,6.6,5.3,4.7,3.3,and2.5kb (not shown); the 8.7-kb band is very similar to the 9.0-kb EcoRI ANX2Ll band (Huebner et al., 1988). For SacI, the annexin III cDNA detects bands of 16.0, 10.5, 4.9, 4.3, 3.1 and 1.4 kb; the 4.3-kb band is very similar to the 4.1-kb ANX2Ll band (Leysens et al.,

6.6

FIG. 4. Genomic Southern blot analysis of human-hamster hybrid panel. Genomic DNA (8 pg) was digested with HindIII; the Southern blot was probed with the annexin III cDNA as described under Materials and Methods.

446

TAIT

a

ET

AL.

40 30 20

g .-

10 j "'"'I'"""

$0

‘ir

1

5 { 4o =

2

3

4

5

6

7

X

30 20 10

0L~&hrrrmtm.mkn&clthdb-dmbanb*cm! a

9

10

11

12

13

14

15

16

17

ia

19

20

21

22

y

b

FIG. 5. Regional location tion on human chromosomes;

of the annexin (B) distribution

III gene to human chromosome of autoradiographic grains

1989). These results suggest that the chromosome 4-specific bands detected by the annexin II cDNA may be due to cross-hybridization with the annexin III gene. DISCUSSION We have shown that annexin III maps to a unique locus on human chromosome 4 at band q21. The results agree for two independent methods (somatic cell hybrid analysis and in situ hybridization). PCR analysis of the hybrid panel provided a clearcut localization to chromosome 4, and this was supplemented by genomic Southern blot analysis of the same panel. The

4 at region on a diagram

4q13-q22. of human

(A) Distribution chromosome

of 138 sites of hybridiza4.

annexin III gene appears to be relatively large (30-40 kb) based on analysis of genomic Southern blots with a cDNA probe. This would be consistent with the properties of the mouse annexin II gene, which contains at least 11 introns and spans at least 22 kb (Amiguet et al., 1990). Although it is possible that a few of the fragments detected in genomic Southern blots with the annexin III cDNA may not be part of the ANX3 locus, the observation of a single major peak by in situ hybridization analysis indicates that most of the detected fragments are part of the ANX3 locus on chromosome 4. Genes for the human annexin protein family reside on several different chromosomes. In addition to

CHROMOSOMAL

MAPPING

ANX3, chromosome 4 contains the gene for annexin V in region q26-q32 (Modi et al., 1989; Tait et al., unpublished). Annexin I has been mapped to a single locus on human chromosome 9 (Huebner et aZ., 1988) and annexin VI to a single locus on human chromosome 5 (Davies et al., 1989). An annexin II (lipocortin II) cDNA probe detects four loci on chromosomes 4,9, 10, and 15 (Huebner et al., 1988). The other known members of the mammalian annexin family (annexins IV, VII, and VIII) have not yet been mapped. The ANX2Ll locus, at 4q21-q31.1 (Huebner et al., 1988), may be identical to the ANX3 locus. Some of the genomic restriction fragments detected by the annexin III cDNA are very similar to the chromosome 4-specific fragments detected by the annexin II cDNA (see Results). The ANX2Ll locus was defined only by hybridization with an annexin II cDNA and has not been shown to be a functional gene (Huebner et al., 1988). However, it is unclear why the annexin II cDNA would cross-hybridize with the annexin III gene, since both theoretical expectation and experimental data (see Results; Amiguet et al., 1990) indicate that annexin cDNAs should not cross-hybridize. All known members of the annexin family are less than 60% identical (Hauptmann et al., 1989). In addition, in the mouse there is a single locus for annexin II on mouse chromosome 9; comparison of linkage groups suggests that the human annexin II gene would be on chromosome 15 (Amiguet et al, 1990), corresponding to the ANX2L4 locus on human chromosome 15 identified by Huebner et al. (1988). This study confirms the value of PCR analysis of somatic cell hybrids for chromosomal mapping studies. As pointed out by Iggo et al. (1989), amplification of intron-containing regions can be performed only on the basis of cDNA sequence data and will often allow discrimination between human and rodent genes because intron size is poorly conserved between species. Likewise, differences in intron size are likely to allow discrimination between related human genes or pseudogenes. Intron-containing PCR products also rule out experimental false positives due to contamination of the PCR reaction with cDNA templates already present in the laboratory. ACKNOWLEDGMENTS We thank Christy Smith for help with some experiments, Don Gibson for oligonucleotide synthesis, Sheyla West for samples of human genomic DNA, and Dr. Jeffrey Murray for providing DNA from the HA(4)A cell line. This work was supported by NIH Grants HL-40801 (to J.F.T.), AG-01751 and March of Dimes l-1019 (to C.M.D.), and GM-37999 and HG-00042 (to A.M.K.). REFERENCES 1.

AMIGUET, P., D’EUSTACHIO, P., KRISTENSEN, T., WETSEL, R. A., SARIS, C. J. M., HUNTER, T., CHAPLIN, D. D., AND

OF

ANNEXIN

III

447

GENE

TACK, B. F. (1990). Structure and chromosome assignment the murine p36 (calpactin I heavy chain) gene. Biochemistry 29: 1226-1232. 2. 3.

of

BURGOYNE, R. D., AND GEISOW, M. J. (1989). The annexin family of calcium-binding proteins. Cell Calcium 10: l-10. BURNS, A. L., MAGENDZO, K., SHIRVAN, A., SRIVASTAVA, M., ROJAS, E., ALIJANI, M. R., AND POLLARD, H. B. (1989). Calcium channel activity of purified human synexin and structure of the human synexin gene. Proc. Natl. Acad. Sci. USA 86: 3798-3802.

4.

CARLOCK, L. R., SMITH, D., AND WASMLITH, J. (1986). Genetic counterselective procedure to isolate interspecific cell hybrids containing single human chromosomes: Construction of cell hybrids and recombinant DNA libraries specific for human chromosomes 3 and 4. Somat. Cell Mol. Genet. 12: 163-174.

5.

CROMPTON, M. (1988). Diversity 1-3.

6.

CRUMPTON, M. J., AND DEDMAN, nology tangle. Nature 346: 212.

7.

DANA, S., AND WASMUTH, J. J. (1982). Linkage of the leuS, emtB, and chr genes on chromosome 5 in humans and expression of human genes encoding protein synthetic components in human-Chinese hamster hybrids. Somat. Cell Genet. 8: 245-264.

8.

DAVIES, A. A., Moss, S. E., CROMPTON, M. R., JONES, T. A., SPURR, N. K., SHEER, D., KOZAK, C., AND CRUMPTON, M. J. (1989). The gene coding for the ~68 calcium-binding protein is localized to bands q32-q34 of human chromosome 5, and to mouse chromosome 11. Hum. Genet. 82: 234-238.

9.

DELANGE, T., SHIRE, L., MYERS, R. M., Cox, D. R., NAYLOR, S. L., KILLARY, A. M., AND VARMUS, H. E. (1990). Structure and variability of human chromosome ends. Mol. Cell. Biol. 10: 518-527.

10.

ERNST, J. D., HOYE, E., BLACKWOOD, R. A., AND JAYE, D. (1990). Purification and characterization of an abundant cytosolic protein from human neutrophils that promotes Ca’+-dependent aggregation of isolated specific granules. J. Clin. Znuest. 86: 1065-1071.

11.

FLAHERTY, M. AND TAIT, J. Measurement static system.

12.

FUNAKOSHI, T., HEIMARK, R. L., HENDRICKSON, L. E., MCMULLEN, B. A., AND FUJIKAWA, K. (1987a). Human placental anticoagulant protein: Isolation and characterization. Biochemistry 26: 5572-5578.

13.

FUNAKOSHI, T., HENDRICKSON, L. E., MCMULLEN, B. A., AND FUJIKAWA, K. (1987b). Primary structure of human placental anticoagulant protein. Biochemistry 26: 8087-8092.

14.

HAIGLER, H. T., FITCH, J. M., JONES, J. M., AND SCHLAEPFER, D. D. (1989). Two lipocortin-like proteins, endonexin II and anchorin CII, may be alternate splices of the same gene. Trends. Biochem. Sci. 14: 48-50. HAUPTMANN, R., MAURER-FOGY, I., KRYSTEK, E., BODO, G., ANDREE, H., AND REUTELINGSPERGER, C. P. M. (1989). Vascular anticoagulant beta: A novel human Ca’+/phospholipid binding protein that inhibits coagulation and phospholipase A, activity. Eur. J. Biochem. 185: 63-71.

15.

16.

R., Moss, S. E., AND in the lipocortin/calpactin

CRUMPTON, family.

J. R. (1990).

Protein

M. J. Cell 55: termi-

J., WEST, S., HEIMARK, R. L., FIJJIKAWA, K., F. (1990). Placental anticoagulant protein-I: in extracellular fluids and cells of the hemoJ. Lab. Clin. Med. 116: 174-181.

HUEBNER, K., CANNIZZARO, L. A., FREY, A., HECHT, HECHT, F., CROCE, C. M., AND WALLNER, B. P. (1988). mosomal localization of the human genes for lipocortin lipocortin II. Oncogene Res. 2: 299-310.

B. K., ChroI and

448

TAIT Xu, W., LANE, D. P., AND SPURR, N. K. mapping of the human gene encoding antigen (~68) by using the polymerase Natl. Acad. Sci. USA 86: 6211-6214.

ET

AL.

17.

IGGO, R., GOUGH, A., (1989). Chromosome the 68-kDa nuclear chain reaction. Proc.

27.

18.

IWASAKI, A., SUDA, M., NAKAO, H., et al. (1987). Structure and expression of cDNA for an inhibitor of blood coagulation isolated from human placenta: A new lipocortin-like protein. J. Biochem. 102: 1261-1273.

19.

JOHNSTON, P. A., PERIN, M. S., REYNOLDS, G. A., WASSERMANN, S. A., AND SUDHOF, T. C. (1990). Two novel annexins from Drosophila melanogaster. J. Biol. Chem. 265: 1138211388.

30.

20.

KAETZEL, M. A., HAZARIKA, Differential tissue expression cium-dependent phospholipid Chem. 264: 14463-14470.

31.

21.

KLEE, C. B. (1988). membrane-) binding

22.

LEYSENS, N. J., NEWKIRK, N. G., AND MURRAY, J. C. (1989). Sac1 and XbaI polymorphisms detected by lipocortin 2A (LPCPA). Nucleic Acids Res. 17: 5417.

23.

MARTH, J. D., DISTECHE, C., PRAVTCHEVA, D., RUDDLE, F., KREBS, E. G., AND PERLMUITER, R. M. (1986). Localization of a lymphocyte-specific protein tyrosine kinase gene (Ick) at a site of frequent chromosomal abnormalities in human lymphomas. Proc. Natl. Acad. Sci. USA 83: 7400-7404.

28.

29.

P., AND DEDMAN, J. R. (1989). of three 35kDa annexin calbinding proteins. J. Biol.

Calcium-dependent phospholipid(and proteins. Biochemistry 27: 6645-6653. 32.

33.

34.

24.

MAURER-FOGY, I., REUTELINGSPERGER, C. P. M., PIETERS, J., BODO, G., STRATOWA, C., AND HAUPTMANN, R. (1988). Cloning and expression of cDNA for human vascular anticoagulant, a Ca’+-dependent phospholipid binding protein. Eur. J. Biochem. 174: 585-592.

25.

MODI, W. S., SEUANEZ, H. N., JAYE, M., HAIGLER, H. J., KAPLAN, R., AND O’BRIEN, S. J. (1989). The human endonexin II (ENXZ) gene is located at 4q28-q32. Cytogenet. Cell Genet. 62: 167-169.

36.

26.

MULLIS, K. B., AND FALOONA, F. A. (1987). of DNA in vitro via a polymerase-catalyzed “Methods in Enzymology” (R. Wu, Ed.), 351, Academic Press, San Diego.

37.

Specific synthesis chain reaction. In Vol. 155, pp. 335-

35.

PEPINSKY, R. B., TIZARD, R., MATTALIANO, R. J., et al. (1988). Five distinct calcium and phospholipid binding proteins share homology with lipocortin I. J. Biol. Chem. 263: 10799-10811. REED, K. C., AND MANN, D. A. (1985). Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res. 13: 7207-7221. REUTELINGSPERGER, C. P. M., HORNSTRA, G., AND HEMKER, H. C. (1985). Isolation and partial purification of a novel anticoagulant from arteries of human umbilical cord. Eur. J. Bio&em. 151: 625-629. REUTELINGSPERGER, C. P. M., KOP, J. M. M., HORNSTRA, G., AND HEMKER, H. C. (1988). Purification andcharacterization of a novel protein from bovine aorta that inhibits coagulation. Eur. J. Biochem. 173: 171-178. Ross, T. S., AND MAJERUS, P. W. (1986). Isolation of D-myoinositol l:2-cyclic phosphate 2-inositolphosphohydrolase from human placenta. J. Biol. Chem. 261: 11119-11123. Ross, T. S., TAIT, J. F., AND MAJERUS, P. W. (1990). Identity of inositol 1,2-cyclic phosphate 2-phosphohydrolase with lipocortin III. Science 248: 605-607. SAIKI, R. K., GELFAND, D. H., STOFFEL, S., et al. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487-491. SAMBROOK, J., FRITSCH, E. F., AND MANIATIS, T. (1989). “Molecular Cloning: A Laboratory Manual,” 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. TAIT, J. F., SAKATA, M., MCMULLEN, B. A., MIAO, C. H., FUNAKOSHI, T., HENDRICKSON, L. E., AND FUJIKAWA, K. (1988). Placental anticoagulant proteins: Isolation and comparative characterization of four members of the lipocortin family. Biochemistry 27: 6268-6276. TAIT, J. F., GIBSON, D., AND FUJIKAWA, K. (1989). Phospholipid binding properties of human placental anticoagulant protein-I, a member of the lipocortin family. J. Biol. Chem. 264: 7944-7949. WALLNER, B. P., MA?TALIANO, R. J., HESSION, C., et al. (1986). Cloning and expression of human lipocortin, a phospholipase A, inhibitor with potential anti-inflammatory activity. Nature 320: 77-81.

Chromosomal localization of the human annexin III (ANX3) gene.

The annexins or lipocortins are a new family of calcium-dependent phospholipid-binding proteins. Annexin III has been previously identified as inosito...
2MB Sizes 0 Downloads 0 Views