We have isolated suppressor mutants that suppress temperature-sensitive colony formation and anucleate cell production of a mukB mutation. A linkage group (smbB) of the suppressor mutations is located in the rne/ams/hmp gene encoding the processing endoribonuclease RNase E. All of the rne (smbB) mutants code for truncated RNase E polypeptides lacking a carboxyl-terminal half. The amount of MukB protein was higher in these rne mutants than that in the rne ؉ strain. These rne mutants grew nearly normally in the mukB ؉ genetic background. The copy number of plasmid pBR322 in these rne mutants was lower than that in the rne ؉ isogenic strain. The results suggest that these rne mutations increase the half-lives of mukB mRNA and RNAI of pBR322, the antisense RNA regulating ColE1-type plasmid replication. We have demonstrated that the wild-type RNase E protein bound to polynucleotide phosphorylase (PNPase) but a truncated RNase E polypeptide lacking the C-terminal half did not. We conclude that the C-terminal half of RNase E is not essential for viability but plays an important role for binding with PNPase. RNase E and PNPase of the multiprotein complex presumably cooperate for effective processing and turnover of specific substrates, such as mRNAs and other RNAs in vivo.RNase E was first defined as a processing endoribonuclease that catalyzes the maturation of 5S rRNA (2,3,17). Mutations in the rne/ams gene have a stabilizing effect on the bulk of mRNAs (4,37,44). This enzyme also cleaves RNAI (27, 33, 52), the antisense RNA regulating ColE1-type plasmid replication (53), and is involved in the processing and turnover of many bacterial and bacteriophage mRNAs (14, 16, 23, 28-30, 35, 42, 46) (for a review, see reference 11). The Escherichia coli rne/ams/hmp gene codes for the RNase E protein of 1,061 amino acids (8). The RNase E protein migrates as a 170-to 180-kDa polypeptide in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (7,12,35). Taraseviciene et al. (50) concluded that the RNA binding domain and catalytic functions overlap in the N-terminal half of the protein. The more precise mapping reported that, while the catalytic function of RNase E is in the N-terminal half, the RNA binding domain is located in the C-terminal half and does not overlap with the catalytic domain (31). It would be more accurate to state that the N-terminal half of Rne (residues 1 to 498) has the catalytic function of RNase E (31, 50) while a nonoverlapping RNA binding activity has been mapped near the start of the C-terminal half (50). RNase E, polynucleotide phosphorylase (PNPase), and 3Ј-to-5Ј exonucleases were found in the same multiprotein complex in E. coli cell extracts (6, 45).The MukB protein of E. coli has an important role in proper partitioning of the replicated sister chromosomes into two daughter cells (40,41,57,59,60). The MukB protein consists of 1,484 amino acid residues (57). MukB has been suggested to be a motor protein essential for chromosome partitioning (41) (for reviews, see references 18 and...

The mukB gene codes for a 177 kDa protein, which might be a candidate for a force-generating enzyme in chromosome positioning in Escherichia coli. The mukB106 mutant produces normal-sized, anucleate cells and shows a temperature-sensitive colony formation. To identify proteins interacting with the MukB protein, we isolated three multicopy suppressors (msmA, msmB, and msmC) to the temperature-sensitive colony formation of the mukB106 mutation. The msmA gene, which could not suppress the production of anucleate cells, was found to be identical to the dksA gene. The msmB and msmC genes suppressed the production of anucleate cells as well as the temperature-sensitive colony formation. However, none of them could suppress both phenotypes in a mukB null mutation. DNA sequencing revealed that the msmB gene was identical to the cspC gene and that the msmC gene had not been described before. A homology search revealed that the amino acid sequences of both MsmB and MsmC possessed high similarity to proteins containing the cold-shock domain, such as CspA of E. coli and the Y-box binding proteins of eukaryotes; this suggests that MsmB and MsmC might be DNA-binding proteins that recognize the CCAAT sequence. Hence, the msmB and msmC genes were renamed cspC and cspE, respectively. Possible mechanisms for suppression of the mukB106 mutation are discussed.

Gastric inhibitory polypeptide (GIP) is a 42-amino acid hormone that stimulates insulin secretion in the presence of glucose. Complementary DNA clones encoding human GIP were isolated from a library prepared with RNA from duodenum. The predicted amino acid sequence indicates that GIP is derived by proteolytic processing of a 153-residue precursor, preproGIP. The GIP moiety is flanked by polypeptide segments of 51 and 60 amino acids at its NH2 and COOH termini, respectively. The former includes a signal peptide of about 21 residues and an NH2-terminal propeptide of 30 amino acids. GIP is released from the precursor by processing at single arginine residues. There is a region of nine amino acids in the COOH-terminal propeptide of the GIP precursor that has partial homology with a portion of chromogranin A as well as pancreastatin.Gastric inhibitory polypeptide (GIP) was first isolated from porcine small intestine (1, 2) on the basis of its ability to inhibit histamine-stimulated gastric acid secretion in the dog stomach (3). The sequences of porcine (4, 5), bovine (6), and human GIP (7) have been determined; each has 42 amino acids, and the sequence is highly conserved. The porcine and bovine peptides differ from the human at two and four sites, respectively. The sequence of GIP indicates that this peptide is a member of a family of structurally related hormones that includes secretin, glucagon, vasoactive intestinal peptide, and growth hormone-releasing factor (8-10). Subsequent studies of the physiological properties of GIP revealed that it was a relatively poor inhibitor of gastric acid secretion (11) but was a potent stimulator of insulin secretion (12, 13). It is able to stimulate insulin secretion at physiological concentration only in the presence of glucose (14,15). Thus, GIP by functioning as a glucose-dependent insulin-releasing peptide could have a role in maintaining glucose homeostasis. Moreover, it might also be of use in treating some diabetics. As a first step in studying the biosynthesis of this potentially important regulator of beta cell function, we have isolated and sequenced cDNA clones encoding the human GIP precursor. This report describes the sequence of these clones and the predicted amino acid sequence of the human protein. Fig. 1 were synthesized by a modification of the triester method (21). Probes I and II were end-labeled at the 5'-terminal end with T4 polynucleotide kinase (Toyobo, Osaka, Japan) and [y-32P]ATP (Amersham; 6000 Ci/mmol; 1 Ci = 37 GBq) and was purified by passing through a Sephadex G-50 (Pharmacia) column. MATERIALS AND METHODSColony Screening. Escherichia coli HB101 was transformed with the recombinant DNA by the calcium-shock procedure (22). Colony hybridization (23) was performed with probe I (14-mer) at 36°C for 18 hr and with probe II (17-mer) at 40°C for 18 hr in 4x NaCl/Cit (lx = 0.15 M NaCI/0.015 M sodium citrate, pH 7) containing 1OX Denhardt's solution (1x = 0.02% polyvinylpyrrolidone/ 0.02% Ficoll/0.2% bovine serum albumin), 0.1% NaDodSO4, 25 ,ug of...

A cDNA clone encoding the human motilin precursor was isolated from an intestinal library using synthetic oligonucleotide probes. The predicted amino acid sequence indicates that the motilin precursor consists of 115 amino acids and includes a 25-residue N-terminal signal peptide followed by the 22-amino-acid motilin sequence and a long, 68-residue C-terminal peptide. The amino acid sequence of human motilin predicted from the cDNA sequence is indentical to its porcine counterpart, which has been determined by protein sequencing. Proteolytic processing of promotilin to motilin occurs at the sequence, Lys-Lys, this being the first reported instance of processing occurring at a pair of Lys residues. In other precursors it occurs at Lys-Arg, Arg-Arg, Arg, or very rarely Lys.cDNA sequence; Motilin; (Human)

The MukB protein is essential for chromosome partitioning in Escherichia coli and consists of 1484 amino acid residues (170 kDa). We have determined the base changes at the mutated sites of the mukB106 mutant and a newly isolated mutant, mukB33. These mutant mukB genes were each found to carry a single base-pair transition which leads to an amino acid substitution; a serine residue at position 33 was changed to phenylalanine in the case of mukB106, and an aspartic acid residue at position 1201 was changed to asparagine in the case of mukB33.