The position of the yeast phosphoglycerate kinase (PGK) gene has been mapped on a 2.95kb Hind III fragment. We have determined the nucleotide sequence of the 5' flanking region and compared this sequence with those from 16 other yeast genes. PGK, like all other yeast genes has an adenine residue at position -3. It has two possible TATA boxes at positions -114 and -152 and a CAAT box at -129. In addition we have defined a structure at position -68 to -39 that is common to all yeast genes that encode an abundant RNA. This structure is a CT-rich block followed, about 10 nucleotides later, by the sequence CAAG.

We have isolated from the hemolymph of immunized larvae of the dipteran insect Phormia terranovae two peptides that are selectively active against Gram-positive bacteria. They are positively charged peptides of 40 residues containing three intramolecular disulfide bridges and differ from one another by only a single amino acid. These peptides are neither functionally nor structurally related to any known insect immune response peptides but show significant homology to microbicidal cationic peptides from mammalian granulocytes (defensins). We propose the name "insect defensins" for these insect antibiotic peptides.

SummaryThe enzyme methylglyoxal synthase (MGS) was partially purified from Escherichia coli extracts, and the amino-terminal sequence of candidate proteins was determined, based on the native protein being a tetramer of about 69 kDa. Database analysis identified an open reading frame in the E. coli genome, YccG, corresponding to a protein of 16.9 kDa. When amplified and expressed from a controlled promoter, it yielded extracts that contained high levels of MGS activity. MGS expressed from the trc promoter accumulated to approximately 20% of total cell protein, representing approximately 900-fold enhanced expression. This caused no detriment during growth on glucose, and the level of methylglyoxal (MG) in the medium rose to only 0.08 mM. High-level expression of MGS severely compromised growth on xylose, arabinose and glycerol. A mutant lacking MGS was constructed, and it grew normally on a range of carbon sources and on low-phosphate medium. However, the mutant failed to produce MG during growth on xylose in the presence of cAMP, and growth was inhibited.

Cadmium-induced metallothioneins from the common sea mussel, Mytilus edulis, were shown to comprise of two groups of isoforms having apparent molecular masses of 10kDa and 20kDa. The 1 0-kDa group was resolved by anion-exchange chromatography into four fractions while the 20-kDa group was resolved into three fractions using this method. After metal removal and Smethylation of the cysteine residues using methyl-p-nitrobenzenesulphonate the complete amino acid sequences were determined. Five isoforms of the 20-kDa group were shown to possess monomeric units consisting of 71 amino acids. These proteins were distinct from the four 72-amino-acid proteins of the 10-kDa group. The FASTA algorithm has been used to compare the degree of similarity between the mussel metallothionein MT-10-IV isoform and other metallothioneins. The mussel MT-10-IV isoform exhibited substantial similarity to other molluscan metallothioneins. Moreover, the mussel metallothionein exhibited more similarity to vertebrate metallothioneins than to those of non-molluscan invertebrates, thus suggesting that the mussel metallothioneins are class I metallothioneins.Despite the discovery of the small cysteine-rich metalbinding-protein metallothionein (MT) more than 30 years ago in equine kidney cortex, the function of this protein is still not fully understood. MT is known to bind metals such as Zn and Cu (which are essential for cell growth and development) and Cd and Hg (which are toxic), which suggests that MT might have more than one function.The search for putative functions for these proteins is exacerbated by the existence of numerous inducers and isoforms. The range of factors which regulate MT synthesis and degradation together with the ubiquity and sequence conservation of MT perhaps suggests that these proteins have a central role in cell metabolism. The proposed functions for MT include the detoxification of heavy metals and the metabolism of essential metals. Zeng et al. (1991) have suggested that thionein may be the active form of the protein and it may be involved in the control of gene expression by virtue of its ability to remove Zn from Zn-finger proteins. Moreover, a tissue-specific isoform of MT in astrocytes (also called growth-inhibitory factor) which inhibits the survival Correspondence to

Injury or injection of live bacteria into third instar larvae of the dipteran insect Phormia terranovae results in the appearance in the haemolymph of at least five groups of heat-stable, more or less basic peptides with antibacterial activity against Escherichia coli. Three of these peptides have been purified. The amino acid sequence has been completely established for one of these and partially (first 40 residues from the N-terminus) for the two others. The sequences show marked homologies indicating that the three peptides belong to a common family. They are not related to other known antibacterial peptides from insects [lysozymes, cecropins (including sarcotoxin I) and attacins]. We propose the name of diptericins for this new family of antibiotic molecules.It is now well established that lepidopteran and dipteran insects respond to a bacterial challenge and also to injury by synthesizing peptides with antibacterial activity (reviewed in Boman and Gotz [l]). Several antibacterial molecules induced in Hyalophora cecropia have been fully or partially characterized : these are the cecropins (3 -5 kDa basic, heatstable peptides) [2], the attacins (20 kDa basic or acidic proteins) [3] and lysozymes [4]. In dipterans, only one induced antibacterial protein has been characterized so far; it is a basic 39-residue molecule named sarcotoxin I, isolated from the blood of injured flesh-fly larvae Sarcophaga peregrina; this molecule shares significant homology with cecropins and is found in at least three forms [5].In the course of an investigation into the cellular and humoral defence reactions of the dipteran Phormia terranovae, a species closely related to Calliphora erythrocephala, we have recently obtained evidence for the appearance in the haemolymph of immunized larvae of a number of heat-stable, basic antibacterial proteins [6, 71. There are at least five of these proteins (or groups of proteins) in the immune haemolymph; none of them corresponds to lysozyme.In the present paper we show that three of these proteins belong to a novel class of antibacterial peptides; in particular, they differ from cecropins, attacins and lysozymes. We propose that they should be named diptericins. We report how, starting with immune plasma of Phormia, we have isolated the induced protein which shows the highest antibacterial activity on Escherichia coli in our assay conditions. It is a basic molecule (PI = 8.5) containing 82 amino acid residues with a relative molecular mass of 8610. We have determined the complete amino acid sequence of this major form (diptericin A) and sufficient N-terminal sequence of two of the minor forms (diptericins B and C) to reveal that there is a family of diptericins of related structure.

The complete amino acid sequence (673 residues plus 15 residues of leader sequence) of human complement component C1s has been determined by nucleotide sequencing of cDNA clones from a human liver library probed with synthetic oligonucleotides. Much of the sequence is supported by independent amino acid sequence information. The cDNA sequence contains an anomalous "intron-like" sequence, including a stop codon, that can be discounted because of the amino acid sequence evidence. The N-terminal chain (422 residues) of C1s, like that of C1r with which it is broadly homologous, contains five domains: domains I and III are homologous to one another and to similar regions in C1r, domain II is homologous to the epidermal growth factor sequence found in C1r and several other proteins, and domains IV and V are homologous to one another and to the 60-residue repeating sequence found in C1r, C2, factor B, C4-binding protein and some apparently unrelated proteins. The sequence of the C-terminal chain (251 residues) agrees with that already established to be the "serine protease" domain of C1s.

We aimed to confirm the ovarian site of gonadotrophin surge-attenuating factor (GnSAF) production and produce granulosa/luteal cell-conditioned medium (G/LCM) containing GnSAF for purification studies. Blue dye affinity chromatography followed by pseudochromatofocusing of G/LCM yielded bioactive fractions at pH 5.74 and 5.77. The former had a major 60-66 kDa band with an internal amino acid sequence of EPQVYVHAP following tryptic digestion. A rat polyclonal antiserum (rPAb) raised against this band completely blocked in-vitro GnSAF bioactivity in human follicular fluid, serum and G/LCM. GnSAF bioactivity was localized to a 64 kDa band of serum-free G/LCM and following 2D gel electrophoresis, one of the spots recognized by Western blotting with the GnSAF rPAb had an N-terminal amino acid sequence of NH-XVPQGNAXXN. Neither amino acid sequence had significant homology with proteins in the human genome database. When ovarian tissues from spontaneously cycling women were cultured under serum-free conditions, neither theca- nor stroma-conditioned media contained GnSAF bioactivity. However, granulosa cell-conditioned medium significantly reduced GnRH-induced LH secretion, an effect that was reversed by incubation with the GnSAF rPAb. In conclusion, we have confirmed that human granulosa cells produce GnSAF within the ovary and have two candidate amino acid sequences for GnSAF. We have also demonstrated that serum-free granulosa cell culture constitutes the method of choice for the characterization of GnSAF since recovery of bioactivity is superior in the presence of fewer serum proteins.

The tryptophan-sensitive 3-deoxy-~-arabino-heptulosonate-7-phosphate (DAHP) synthases from Streptomyces coelicolor A3(2), Streptomyces rimosus and Neurospora crassa have been purified to homogeneity. All three enzymes have a subunit M, of 54000. The S. coelicolor DAHP synthase was physically and kinetically characterized and the N-terminal amino acid sequence was obtained. The N-terminal amino acid sequence could not be obtained for the enzymes from 5. rimosus and N. crassa, their N-termini apparently being blocked. However, following proteolytic digestion, internal amino acid sequences were obtained from both enzymes. A comparison with the known DAHP synthase sequences indicated that these DAHP synthases are unrelated to other microbial DAHP synthase sequences but are similar to plant DAHP synthases. Up until now, two distinct classes of DAHP synthase have been described, one comprising exclusively enzymes from plants, the other restricted t o enzymes from micro-organisms. These studies indicate that the class containing the plant DAHP synthases also contains enzymes from a microbial eukaryote and from several bacteria.