Glycosylated and unglycosylated recombinant-derived human stem cell factors are dimeric and have extensive regular secondary structure.

We have recently described the identification, isolation, and characterization of a factor, termed stem cell factor (SCF), which acts on primitive hematopoietic progenitors of the marrow. A soluble form of the factor was isolated from the conditioned medium of a rat cell line (Zsebo, K. M., Wypych, J., McNiece, I. K., Lu, H. S., Smith, K. A., Karkare, S. B., Sachdev, R. K., Yuschenkoff, V. N., Birkett, N. C., Williams, L. R., Satyagal, V. N., Tung, W., Bosselman, R. A., Mendiaz, E. A., and Langley, K. E. (1990) Cell 63, 195-201) and rat and human cDNAs have been cloned (Martin, F. H., Suggs, S. V., Langley, K. E., Lu, H. S., Ting, J., Okino, K. H., Morris, C. F., McNiece, I. K., Jacobsen, F. W., Mendiaz, E. A., Birkett, N. C., Smith, K. A., Johnson, M. J., Parker, V. P., Flores, J. C., Patel, A. C., Fisher, E. F., Erjavec, H. O., Herrera, C. J., Wypych, J., Sachdev, R. K., Pope, J. A., Leslie, I., Wen, D., Lin, C.-H., Cupples, R. L., and Zsebo, K. M. (1990) Cell 63, 203-211). The cDNAs encode amino acids C-terminal to those found in the isolated natural form, including a putative transmembrane domain. This paper describes the structural characterization of soluble forms of recombinant human SCF purified from Escherichia coli (unglycosylated) and from Chinese hamster ovary (CHO) cells (glycosylated). Fluorescence emission spectra indicate that the single Trp residue is present in a hydrophobic environment. Circular dichroism and infrared spectroscopy indicate considerable secondary structure, including both alpha-helix and beta-sheet. Molecular weight determinations by sedimentation equilibrium show that the molecules are dimeric (noncovalently associated), and gel filtration analyses are consistent with this conclusion. The CHO cell-derived SCF is about 30% carbohydrate by weight, with both N-linked and O-linked sugar. The presence or absence of the carbohydrate does not influence the results of the various structural analyses.     

The complete amino acid sequence of coagulogen isolated from Southeast Asian horseshoe crab, Carcinoscorpius rotundicauda

The complete amino acid sequence of coagulogen purified from the hemocytes of the horseshoe crab Carcinoscorpius rotundicauda was determined by characterization of the NH2-terminal sequence and the peptides generated after digestion of the protein with lysyl endopeptidase, Staphylococcal aureus protease V8 and trypsin. Upon sequencing the peptides by the automated Edman method, the following sequence was obtained: A D T N A P L C L C D E P G I L G R N Q L V T P E V K E K I E K A V E A V A E E S G V S G R G F S L F S H H P V F R E C G K Y E C R T V R P E H T R C Y N F P P F V H F T S E C P V S T R D C E P V F G Y T V A G E F R V I V Q A P R A G F R Q C V W Q H K C R Y G S N N C G F S G R C T Q Q R S V V R L V T Y N L E K D G F L C E S F R T C C G C P C R N Y Carcinoscorpius coagulogen consists of a single polypeptide chain with a total of 175 amino acid residues and a calculated molecular weight of 19,675. The secondary structure calculated by the method of Chou and Fasman reveals the presence of an alpha-helix region in the peptide C segment (residue Nos. 19 to 46), which is released during the proteolytic conversion of coagulogen to coagulin gel. The beta-sheet structure and the 16 half-cystines found in the molecule appear to yield a compact protein stable to acid and heat. The amino acid sequences of coagulogen of four species of limulus have been compared and the interspecies evolutionary differences are discussed.     

Disulfide pairing of the recombinant kringle-2 domain of tissue plasminogen activator produced in Escherichia coli.

The kringle-2 domain of tissue plasminogen activator, cloned and expressed in Escherichia coli (Wilhelm, O.G., Jaskunas, S.R., Vlahos, C.J., and Bang, N.U. (1990) J. Biol. Chem. 265, 14606-14611), was internally radiolabeled using [35S]methionine-cysteine. Following refolding and isolation, the labeled polypeptide was further purified by reverse-phase high performance liquid chromatography. The purified kringle-2 domain was digested with thermolysin, and the resulting peptides were purified by high performance liquid chromatography. Five major peptides containing 35S were obtained. Amino acid sequence analysis showed that these peptides represented various cleavage products containing one or more of the following disulfides: Cys180-Cys261, Cys201-Cys243, Cys232-Cys256 (sequence numbering based on Pennica et al. (Pennica, D., Holmes, W.E., Kohr, W.J., Hakins, R.N., Vehar, G. A., Ward, C.A., Bennett, W.F., Yelverton E., Seeburg, P.H., Heynecker, H.L., Goeddel, E.V., and Collen, D. (1983) Nature 301, 214-221)). These results confirm that the refolding methodology used produced kringle-2 with the predicted disulfide linkage and, thus, yielded material suitable for structural and functional studies.     

The complete amino acid sequence of the low molecular weight cytosolic acid phosphatase

This paper presents the complete amino acid sequence of the low molecular weight acid phosphatase from bovine liver. This isoenzyme of the acid phosphatase family is located in the cytosol, is not inhibited by L-(+)-tartrate and fluoride ions, but is inhibited by sulfhydryl reagents. The enzyme consists of 157 amino acid residues, has an acetylated NH2 terminus, and has arginine as the COOH-terminal residue. All 8 half-cystine residues are in the free thiol form. The molecular weight calculated from the sequence is 17,953. The sequence was determined by characterizing the peptides purified by reverse-phase high performance liquid chromatography from tryptic, thermolytic, peptic, Staphylococcus aureus protease, and chymotryptic digests of the carboxymethylated protein. No sequence homologies were found with the two known acylphosphatase isoenzymes or the metalloproteins porcine uteroferrin and purple acid phosphatase from bovine spleen (both of which have acid phosphatase activity). Two half-cystines at or near the active site were identified through the reaction of the enzyme with [14C] iodoacetate in the presence or in the absence of a competitive inhibitor (i.e. inorganic phosphate). Ac-A E Q V T K S V L F V C L G N I C R S P I A E A V F R K L V T D Q N I S D N W V I D S G A V S D W N V G R S P N P R A V S C L R N H G I N T A H K A R Q V T K E D F V T F D Y I L C M D E S N L R D L N R K S N Q V K N C R A K I E L L G S Y D P Q K Q L I I E D P Y Y G N D A D F E T V Y Q Q C V R C C R A F L E K V R-OH.     

Modulation of the catalytic activity of neutrophil collagenase MMP-8 on bovine collagen I. Role of the activation cleavage and of the hemopexin-like domain

The cleavage of bovine collagen I by neutrophil collagenase MMP-8 has been followed at pH 7.4, 37 degrees C. The behavior of the whole enzyme molecule (whMMP-8), displaying both the catalytic domain and the hemopexin-like domain, has been compared under the same experimental conditions with that of the catalytic domain only. The main observation is that whMMP-8 cleaves bovine collagen I only at a single specific site, as already reported by many others (Mallya, S. K., Mookhtiar, K. A., Gao, Y., Brew, K., Dioszegi, M., Birkedal-Hansen, H., and van Wart, H. E. (1990) Biochemistry 29, 10628-10634; Kn?uper, V., Osthues, A., DeClerk, Y. A., Langley, K. A., Bl?ser, J., and Tschesche, H. (1993) Biochem. J. 291, 847-854; Marini, S., Fasciglione, G. F., De Sanctis, G., D'Alessio, S., Politi, V., and Coletta, M. (2000) J. Biol. Chem. 275, 18657-18663), whereas the catalytic domain lacks this specificity and cleaves the collagen molecule at multiple sites. Furthermore, a meaningful difference is observed for the cleavage features displayed by two forms of the catalytic domain, which differ for the N terminus resulting from the activation process (i.e. the former Met(80) of the proenzyme (MetMMP-8) and the former Phe(79) of the proenzyme (PheMMP-8)). Thus, the PheMMP-8 species is characterized by a much faster k(cat)/K(m), fully attributable to a lower K(m), suggesting that the conformation of the catalytic domain, induced by the insertion of this N-terminal residue in a specific pocket (Reinemer, P., Grams, F., Huber, R., Kleine, T., Schnierer, S., Piper, M., Tschesche, H., and Bode, W. (1994) FEBS Lett. 338, 227-233), brings about a better, although less discriminatory, recognition process of cleavage site(s) on bovine collagen I.     

REVIEWS

Grazing in Terrestrial and Marine Environments (British Ecological Society Symposium No. 4). Edited by D. J. C risp
Fundamentals of Botany Series . Edited by W. A. J ensen and L. G. K avaljian
Multivariate Statistical Analysis for Biologists . By H ilary S eal
Differenciation des Cellules Sexuelles et Eécondation chez les Cryptogames (Protoplasmatologia , Vol. VII/3b). By B. V azart
Allgemeine Botanik . By W. N ultsch
Piante Medicinali, Chimica, Farmacologia e Terapia . Vol. II. By R. B enigni , C. C apra and P. E. C attorini
Thermophilic Fungi . By D onald G. C ooney and R alph E merson
The Genera of Flowering Flants (Angiospermae). Based Principally on the Genera Plantarum of G. Bentham and J. D. Hooker. Dicotyledones . vol. 1. By J. H utchinson
Algal Cultures and Phytoplankton Ecology . By G. E. F ogg
Flora Europaea, Vol . 1. Eycopodiaceae to Plantanaceae . Edited by T. G. T utin , V. H. H eywood , N. A. B urges , D. H. V alentine , S. M. W alters and D. A. W ebb
The Biology of the Algae . By F. E. R ound
Flora of the British Isles Illustrations . Part 4. Monocotyledons . Drawings by S ybil J. R oles
Plants and the Ecosystem . By W. D. B illings . (Fundamentals of Botany Series, edited by W. A. J ensen and L. G. K avaljian
Essays on Crop Evolution . Edited by S ir J oseph H utchinson     

Mosaic evolution of prepropancreatic polypeptide

Pancreatic polypeptide, a 36-amino acid peptide hormone, is synthesized in pancreatic islets of Langerhans and acts as a regulator of pancreatic and gastrointestinal functions. We isolated cDNA clones encoding rat pancreatic polypeptide precursor from an islet cDNA library and determined their nucleic acid sequences. Rat pancreatic polypeptide was found to be flanked on the amino terminus by a putative signal peptide and on the carboxyl terminus by Gly-Lys-Arg followed by a 30-amino acid peptide. Nucleotide and amino acid sequences of the signal peptide and the pancreatic polypeptide of the rat were highly homologous to those of the human (Boel, E., Schwartz, T. W., Norris, K. E., and Fill, N. P. (1984) EMBO J. 3, 909-912). On the other hand, the rat carboxyl-terminal peptide differed markedly from the corresponding domain of the human precursor and did not contain any sequence similar to the icosapeptide, which has so far been known to be a second stable product from mammalian pancreatic polypeptide precursors (Schwartz, T. W., Hansen, H. F., Hakanson, R., Sundler, F., and Tager, H. S. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 708-712). The mosaicism of sequence conservation and divergence in prepropancreatic polypeptides may be a unique example in the evolution of prohormones.     

REVIEWS

Book reviewed in this article
Mycoplasma Diseases of Trees and Shrubs. Ed. by K. M aramorosch and S. P. R aychaudhuhi .
Alpine Rasengesellschaften auf Silikatgestein bei Davos. By L uca V etterli .
The Rust Fungi. Ed. by K. J. S cott and A. K. C hakravorty .
Environment and Plant Ecology. By J. R. E thehington .
Cryptogams. By K. E sser
Nitrogen fixation. Volume 1: Ecology. Ed. by W. J. B roughton .
Agricultural Plants. By R. H. M. L anger and G. D. H ill
Decomposer Basidiomycetes: their Biology and Ecology. Ed. by J. C. F rankland , J. N. H edger and M. J. S wift .
Cell Walls'81: Proceeding of the Second Cell Wall Meeting held in Gottingen, April 8–11, 1981. Ed. by D. G. R obinson and H. Q uader .     

cDNA sequence and homologies of the "57-kDa" nucleotide-binding subunit of the vacuolar ATPase from Arabidopsis

Functional and structural similarities among a wide variety of endomembrane H+-ATPases suggest that they form a distinct class with a common origin. Immunological studies (Manolson, M. F., Percy, J. M., Apps, D. K., Xie, X. S., Stone, D. K., and Poole, R. J. (1987) in Proceedings of the Membrane Protein Symposium (Goheen, S. C., ed) pp. 427-434, Bio-Rad, Richmond, CA, and M. F. Manolson, J. M. Percy, D. K. Apps, X. S. Xie, D. K. Stone, M. Harrison, D. J. Clarke, R. J. Poole, unpublished data) support this idea and suggest an evolutionary relationship between the endomembrane and F0F1 ATPases. Further examination of relationships necessitates comparison of protein/nucleic acid sequence data. To this end, we have cloned and sequenced the cDNA encoding the 57-kDa polypeptide of the Arabidopsis vacuolar membrane H+-ATPase. To our knowledge, this is the first report of the sequence of a "57-kDa" subunit for plant or animal endomembrane H+-ATPase. This cDNA encodes a hydrophilic polypeptide containing a putative ATP binding site. Lack of a secretion signal sequence suggests it is not processed through the endoplasmic reticulum but translated on cytosolic ribosomes. Comparison of protein sequences shows the 57-kDa subunit from Arabidopsis to be nearly identical with the corresponding subunit in Neurospora vacuolar membrane H+-ATPase, very similar to the beta subunit of the archaebacterium Sulfolobus, and slightly, but nevertheless significantly, homologous to the alpha and beta subunits of the F0F1-ATPases. These results suggest that these different classes of ATPases have evolved from a common ancestor.     

Bovine factor VII. Its purification and complete amino acid sequence

A modified method for purification of blood clotting factor VII from bovine plasma was developed, and its complete amino acid sequence was established. The isolated factor VII was activated with factor XIIa, and the resulting two-chain factor VII (factor VIIa) was reduced and S-pyridylethylated or S-aminoethylated. The amino acid sequences of the S-alkylated heavy and light chains were determined by sequencing the fragments obtained from enzymatic and chemical cleavages. Fast atom bombardment mass spectrometry was also used to establish the COOH-terminal sequence of the heavy chain. The light chain consists of 152 residues with one carbohydrate chain at Asn145, and 11 gamma-carboxyglutamic acid residues are found within the NH2-terminal 35 residues. The light chain contains 0.2-0.3 mol of beta-hydroxyaspartic acid/mol of protein, indicating that an aspartic acid residue in bovine factor VII is incompletely hydroxylated. Moreover, a pentapeptide, Ala-Ser*-Ser-Pro-Cys (positions 51-55), isolated from an enzymatic digest of the light chain, contained an unknown serine derivative, but its structure is still unclear. On the other hand, the heavy chain is composed of 255 residues and one asparagine-linked carbohydrate chain at Asn203. Bovine factor VII, with a total of 407 residues, has 71% sequence identity with the human molecule (406 residues) predicted from the cDNA sequence (Hagen, F. S., Gray, C. L., O'Hara, P., Grant, F. J., Saari, G. C., Woodbury, R. G., Hart, C. E., Insley, M., Kisiel, W., Kurachi, K., and Davie, E. W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2412-2416).     

Books

Books reviewed in this article:
B aker . K. 1993. Identification Guide to European Non-passerines.
B arnard . C., G ilbert , F. & M c G regor
B askett , T.S., S ayre . M.W., T omlinson , R.E. & M irarchi .
B ezzel . E. 1993. Kompendium der Vögel Mitteleuropas.
B right , M. 1993. The Private Life of Birds.
C ook , M. 1992. The Birds of Moray and Nairn.
D avison . G.W.H. 1992. Birds of Mount Kinabalu. Borneo.
E rritzoe . J. 1993. The Buds of CITES and How to Identify Them.
F arner , D.S., K ing , J.R. & P arkes , K.C.
G ibbons , D.W., R eid , J.B. & C hapman . R.A. (eds). 1993. The New Atlas of Breeding Birds in Britain and Ireland.
H illman , J.C.
H uxley . E.
J ackson . C.E. 1993. Great Bird Paintings of the World.
J ohnsgard . P.A. 1993. Cormorants, Darters and Pelicans of the World.
M adge . S. & B urn , H. 1994. Crows and Jays. A Guide to the Crows, Jays and Magpies of the World.
N icolai . B. (ed.).
P ower , D.M. (ed.).
P riklonskiy . S.G. (ed.).
R alph . R. 1993. William MacGillivray.
R obinson , D. & C hapman , A.
S harp . P.J. 1993. Avian Endocrinology.
S mith , K.W., D fe , C.W., F earnside . J.D., F letcher , E.W. & S mith , R.N.
S olomon . D. & W illiams , J.
S ørensen , S., B loch . D. & L angvad . S.
Z immerman , J.L.     

The hereditary hemochromatosis protein, HFE, specifically regulates transferrin-mediated iron uptake in HeLa cells

HFE is the protein product of the gene mutated in the autosomal recessive disease hereditary hemochromatosis (Feder, J. N., Gnirke, A., Thomas, W., Tsuchihashi, Z., Ruddy, D. A., Basava, A., Dormishian, F., Domingo, R. J., Ellis, M. C., Fullan, A., Hinton, L. M., Jones, N. L., Kimmel, B. E., Kronmal, G. S., Lauer, P., Lee, V. K., Loeb, D. B., Mapa, F. A., McClelland, E., Meyer, N. C., Mintier, G. A., Moeller, N., Moore, T., Morikang, E., Prasss, C. E., Quintana, L., Starnes, S. M., Schatzman, R. C., Brunke, K. J., Drayna, D. T., Risch, N. J., Bacon, B. R., and Wolff, R. R. (1996) Nat. Genet. 13, 399-408). At the cell surface, HFE complexes with transferrin receptor (TfR), increasing the dissociation constant of transferrin (Tf) for its receptor 10-fold (Gross, C. N., Irrinki, A., Feder, J. N., and Enns, C. A. (1998) J. Biol. Chem. 273, 22068-22074; Feder, J. N., Penny, D. M., Irrinki, A., Lee, V. K., Lebron, J. A., Watson, N. , Tsuchihashi, Z., Sigal, E., Bjorkman, P. J., and Schatzman, R. C. (1998) Proc. Natl. Acad. Sci. U S A 95, 1472-1477). HFE does not remain at the cell surface, but traffics with TfR to Tf-positive internal compartments (Gross et al., 1998). Using a HeLa cell line in which the expression of HFE is controlled by tetracycline, we show that the expression of HFE reduces 55Fe uptake from Tf by 33% but does not affect the endocytic or exocytic rates of TfR cycling. Therefore, HFE appears to reduce cellular acquisition of iron from Tf within endocytic compartments. HFE specifically reduces iron uptake from Tf, as non-Tf-mediated iron uptake from Fe-nitrilotriacetic acid is not altered. These results explain the decreased ferritin levels seen in our HeLa cell system and demonstrate the specific control of HFE over the Tf-mediated pathway of iron uptake. These results also have implications for the understanding of cellular iron homeostasis in organs such as the liver, pancreas, heart, and spleen that are iron loaded in hereditary hemochromatotic individuals lacking functional HFE.     

BOOK REVIEW SECTION

Book Reviewed in this article:
Canning, Elizabeth U., ed. 1981. Parasitological Topics: a Presentation Volume to P. C. C. Garnham F.R.S. on the Occasion of His 80th Birthday 1981
Krylov, M. V. 1981. Piroplazmidy. [Piroplasms.]
Baker, J. R. 1982. The Biology of Parasitic Protozoa
Barriga, Omar O. 1981. The Immunology of Parasitic Infections: a Handbook for Physicians, Veterinarians, and Biologists
Beyer, T. V., Bezukladnikova, N. A., Galuzo, I. G., Konovalova, S. I. & Pak, S. M., eds. 1979. Toksoplasmidy. [The Toxoplasmids
Geltzer, Ya. G., Korganova, G. A., Mavlyanova, M. I. & Nikolyuk, V. I., eds. 1980. Pochvennye Prosteyshie. [The Soil Protozoa.] (Protozoologiya
Beyer, T. V., Kazakova, I. I., Lakhonina, G. M., Roigas, E. M. & Teras, J. H., eds. 1981. Vzaimootnosheniya Prosteyshikh s Virusami. [The Interaction between Protozoa and Viruses.] (Protozoologiya
Ogimoto, Keiji & Imai, Soichi 1981 Allas of Rumen Microbiology
Long, Peter L., ed. 1982. The Biology of the Coccidia
Lloyd, David, Poole, Robert & Edwards, Steven W. 1982. The Cell Division Cycle: Temporal Organization and Control of Cellular Growth and Reproduction
Frederick, J. F., ed. 1981. Origins and Evolution of Eukaryotic Intracellular Organelles. [Ann. N.Y. Acad. Sci.
Hayat, M. A. 1981. Fixation for Electron Microscopy
Buetow, D. E., ed. 1982. The Biology of Euglena.
Ogden, C. G. & Hedley, R. H. 1980. An Atlas of Freshwater Testate Amoebae
Parker, S. P., ed. 1982. Synopsis and Classification of Living Organisms
Margulis, L. & Schwartz, K. V. 1982. Five Kingdoms: an Illustrated Guide to the Phyla of Life on Earth
Cairns, J., Jr., ed. 1982. Artificial Substrates
Curds, C. R. 1982. British and Other Freshwater Ciliated Protozoa     

Identification of a chloroplast-encoded 9-kDa polypeptide as a 2[4Fe-4S] protein carrying centers A and B of photosystem I

An improved procedure is reported for large-scale preparation of photosystem I (PS-I) vesicles from thylakoid membranes of barley (Hordeum vulgare L.). The PS-I vesicles contain polypeptides of molecular masses 82, 18, 16, 14, and 9 kDa in an apparent molar ratio of 4:2:2:1:2. The 18-, 16-, and 9-kDa polypeptides were purified to homogeneity after exposure of the PS-I vesicles to chaotropic agents. The isolated 9-kDa polypeptide binds 65-70% of the zero-valence sulfur of denatured PS-I vesicles, and the remaining 30-35% is bound to P700-chlorophyll a-protein 1. The N-terminal amino acid sequence (29 residues) of the 9-kDa polypeptide was determined. Comparison with the nucleotide sequence of the chloroplast genome of Marchantia polymorpha (Ohyama, K., Fukuzawa, H., Kohchi, T., Shirai, H., Sano, T., Sano, S., Umesono, K., Shiki, Y., Takeuchi, M., Chang, Z., Aota, S.-i., Inokuchi, H., and Ozeki, H. (1986) Nature 322, 572-574) and of Nicotiana tabacum (Shinozaki, K., Ohme, M., Tanaka, M., Wakasugi, T., Hayashida, N., Matsubayashi, T., Zaita, W., Chunwongse, J., Obokata, J., Yamaguchi-Shinozaki, K., Ohto, C., Torazawa, K., Meng, B. Y., Sugita, M., Deno, H., Kamogashira, T., Yamada, K., Kusuda, J., Takaiwa, F., Kato, A., Tohdoh, N., Shimada, H., and Sugiura, M. (1986) EMBO J. 5, 2043-2049) identified the chloroplast gene encoding the 9-kDa polypeptide. We designate this gene psaC. The complete amino acid sequence deduced from the psaC gene identifies the 9-kDa PS-I polypeptide as a 2[4Fe-4S] protein. Since P700-chlorophyll a-protein 1 carries center X, the 9-kDa polypeptide carries centers A and B. A hydropathy plot permits specific identification of the cysteine residues which coordinate centers A and B, respectively. Except for the loss of the N-terminal methionine residue, the primary translation product of the psaC gene is not proteolytically processed. P700-chlorophyll a-protein 1 binds 4 iron atoms and 4 molecules of acid-labile sulfide/molecule of P700. Each of the two apoproteins of P700-chlorophyll a-protein 1 contains the sequence Phe-Pro-Cys-Asp-Gly-Pro-Gly-Arg-Gly-Gly-Thr-Cys (Fish, L. E., Kück, U., and Bogorad, L. (1985) J. Biol. Chem. 260, 1413-1421). The stoichiometry of the component polypeptides of PS-I indicates the presence of four copies of this sequence per molecule of P700. Center X may be composed of two [2Fe-2S] centers bound to the 8 cysteine residues contained in these four segments.     

Genes encoding the core proteins of adenovirus type 2

The nucleotide sequence of the HindIII-D fragment of adenovirus type 2 has been determined. The sequence, which is located between coordinates 41.8 and 51.0, covers most of the L2 cotermination family. It includes three major open translational reading frames encoding the carboxyl-terminal part of the penton base as well as the major core polypeptides V and VII. An additional minor open translational reading frame encoding a highly basic polypeptide was detected in the sequence. The L2 region has a very compact organization with very short distances between the different genes, although no overlapping coding sequences were found. The predicted amino acid sequences of core proteins V and VII reveal that they are highly basic proteins and polypeptide VII resembles the arginine-rich H4 histones in its amino acid composition, but no striking similarities are apparent at the amino acid sequence level. The candidate polypeptide encoded by the newly discovered translational reading frame contains 29% basic residues and includes a hypothetical recognition sequence for the adenovirus-encoded endopeptidase. In conjunction with previously published sequences and those reported in accompanying papers (Akusj?rvi, G., Alestr?m, P., Pettersson, M., Lager, M., J?rnvall, H., and Pettersson, U. (1984) J. Biol. Chem. 259, 13976-13979; Roberts, R. J., O'Neill, K. E., and Yen, C. E. (1984) J. Biol. Chem. 259, 13965-13975) a complete sequence can now be reconstructed for the 35,937-base pairs adenovirus type 2 genome.     

Structure of murine complement component C3. II. Nucleotide sequence of cloned complementary DNA coding for the alpha chain

From the inserts of two recombinant plasmids isolated from a murine liver cDNA library, the nucleotide sequence coding for the C3 alpha chain was obtained and the corresponding amino acid sequence was derived. The alpha chain portion of the mRNA is 2979 nucleotides long, specifying a polypeptide of 993 amino acids. The molecular weight of the alpha chain, in the absence of carbohydrate, was calculated from the sequence data to be 112,933. Two possible carbohydrate attachment sites were predicted at residues 269 (Asn) and 947 (Asn). In addition, the positions for two putative factor I cleavage sites were predicted from a comparison with the cleavage sites in the human C3 alpha chain. The C3 alpha chain contains 24 cysteine residues, 10 of these clustered in the C-terminal 175 amino acids of the alpha chain. Together with the accompanying report (Lundwall, A, Wetsel, R.A., Domdey, H., Tack, B.F., and Fey, G.H. (1984) J. Biol. Chem. 259, 13851-13856), this study completes the nucleotide and amino acid structure of the murine precursor prepro-C3 molecule.     

REVIEWS

The Morphology of Gymnosperms . By K. R. S porne
The Rice Blast Disease
An Account of the Herbaria of the Department of Botany in the University of Oxford . By H ermia N. C lokie
Chemical Microbiology . By A nthony H. R ose
Pflanzenschutzmittel-Rückstände . By H ans M aier -B ode
The State and Movement of Water in Living Organisms . Edited by G. E. F ogg
Teach Yourself Genetics . By R. J. B erry
Floral Biology . By M ary S. P ercival
Vascular Plants: Form and Function . By F. B. S alisbury and R. V. P arke
Nonvascular Plants: Form and Function . By W. T. D oyle
The Molds and Man . By C lyde M. C hristensen
Spore Liberation . By C. T. I ngold     

Book Reviews

STUDIES IN BIOLOGY, NO. 37. PHOTOSYNTHESIS. 3rd Edition. By D.O. H all & K.K. R ao
STUDIES IN BIOLOGY, NO. 48. PLANTS AND MINERAL SALTS. 2nd Edition. By J.F. S utcliffe & D.A. B aker
THE STUDY OF PLANT STRUCTURE: PRINCIPLES AND SELECTED METHODS. By T.P. O'B rien & M.E. McC utly
ADAPTATION OF PLANTS TO WATER AND HIGH TEMPERATURE STRESS. Edited by N.C. T urner & P.J. K ramer
The Biometry of Plant Growth . By D.R. C auston & J.C. V enus     

ElaC encodes a novel binuclear zinc phosphodiesterase

ElaC is a widespread gene found in eubacteria, archaebacteria, and mammals with a highly conserved sequence. Two human ElaC variants were recently associated with cancer (Tavtigian, S. V., Simard, J., Teng, D. H., Abtin, V., Baumgard, M., Beck, A., Camp, N. J., Carillo, A. R., Chen, Y., Dayananth, P., Desrochers, M., Dumont, M., Farnham, J. M., Frank, D., Frye, C., Ghaffari, S., Gupte, J. S., Hu, R., Iliev, D., Janecki, T., Kort, E. N., Laity, K. E., Leavitt, A., Leblanc, G., McArthur-Morrison, J., Pederson, A., Penn, B., Peterson, K. T., Reid, J. E., Richards, S., Schroeder, M., Smith, R., Snyder, S. C., Swedlund, B., Swensen, J., Thomas, A., Tranchant, M., Woodland, A. M., Labrie, F., Skolnick, M. H., Neuhausen, S., Rommens, J., and Cannon-Albright, L. A. (2001) Nat. Genet. 27, 172-180; Yanaihara, N., Kohno, T., Takakura, S., Takei, K., Otsuka, A., Sunaga, N., Takahashi, M., Yamazaki, M., Tashiro, H., Fukuzumi, Y., Fujimori, Y., Hagiwara, K., Tanaka, T., and Yokota, J. (2001) Genomics 72, 169-179). Analysis of the primary sequence indicates homology to an arylsulfatase and predicts a metallo-beta-lactamase fold. At present, no ElaC gene product has been investigated. We cloned the Escherichia coli ElaC gene and purified the recombinant gene product. An enzymatic analysis showed that ElaC does not encode an arylsulfatase but rather encodes a phosphodiesterase that hydrolyzes bis(p-nitrophenyl)phosphate with a k(cat) of 59 s(-1) and K' of 4 mm. Kinetic analysis of the dimeric enzyme revealed positive cooperativity for the substrate bis(p-nitrophenyl)phosphate with a Hill coefficient of 1.6, whereas hydrolysis of the substrate thymidine-5'-p-nitrophenyl phosphate followed Michaelis-Menten kinetics. Furthermore, the enzyme is capable of binding two zinc or two iron ions. However, it displays phosphodiesterase activity only in the zinc form. The metal environment characterized by zinc K-edge x-ray absorption spectroscopy was modeled with two histidine residues, one carboxylate group, and 1.5 oxygen atoms. This corresponds to the coordination found in other metallo-beta-lactamase domain proteins. Phosphodiesterase activity is strongly dependent on the presence of zinc. These results identify the currently unassigned gene product ElaC to be a novel binuclear zinc phosphodiesterase.     

Disruption of disulfide bonds is responsible for impaired secretion in human complement factor H deficiency

Factor H, a secretory glycoprotein composed of 20 short consensus repeat modules, is an inhibitor of the complement system. Previous studies of inherited factor H deficiency revealed single amino acid substitutions at conserved cysteine residues, on one allele arginine for cysteine 518 (C518R) and on the other tyrosine for cysteine 941 (C941Y) (Ault, B. H., Schmidt, B. Z., Fowler, N. L., Kashtan, C. E., Ahmed, A. E., Vogt, B. A., and Colten, H. R. (1997) J. Biol. Chem. 272, 25168-25175). To ascertain if the phenotype, impaired secretion of factor H, is due to the C518R substitution or the C941Y substitution and to ascertain the mechanism by which secretion is impaired, we studied COS-1 and HepG2 cells transfected with wild type and several mutant factor H molecules. The results showed markedly impaired secretion of both C518R and C941Y factor H as well as that of factor H molecules bearing alanine or arginine substitutions at the Cys518-Cys546 disulfide bond (C518A, C546A, C546R, C518A-C546A). In each case, mutant factor H was retained in the endoplasmic reticulum and degraded relatively slowly as compared with most other mutant secretory and membrane proteins that are retained in the endoplasmic reticulum. These data indicate that impaired secretion of the naturally occurring C518R and C941Y mutant factor H proteins is due to disruption of framework-specific disulfide bonds in factor H short consensus repeat modules.