Covalent attachment of myristic acid to pp6o0-sc, the transforming protein of Rous sarcoma virus, was studied in a cell-free system. Using a synthetic peptide containing the first 11 amino acids of the mature pp60 V-src polypeptide sequence as a substrate, we probed lysates from a variety of cells and tissues for N-myristyl transferase (NMT) activity. Nearly every eucaryotic cell type tested contained NMT, including avian, mammalian, insect, and plant cells. Since NMT activity was detected in rabbit reticulocyte lysates, we took advantage of the translational capability of these lysates to determine the precise point during translation at which myristate is attached to pp6o0-src. src mRNA, transcribed from cloned v-src DNA, was translated in reticulocyte lysates which had been depleted of endogenous myristate. Addition of [3H]myristate to lysates 10 min after the start of synchronized translation resulted in a dramatic decrease in the incorporation of radiolabeled myristate into pp60v-src polypeptide chains. These results imply that although myristate can be attached posttranslationally to synthetic peptide substrates, myristylation in vivo is apparently a very early cotranslational event which occurs before the first 100 amino acids of the nascent polypeptide chain are polymerized.Covalent attachment of the 14-carbon fatty acid, myristic acid, has been shown to occur on a wide variety of viral and cellular proteins (11,18,22,23,35,41). One of the bestcharacterized myristylated proteins is pp6O-srC, the transforming protein of Rous sarcoma virus (RSV; 6, 14). Studies of numerous point and deletion mutants of pp6O-src which lack the myristate attachment site have established that myristylation is a necessary event for directing pp60v-sr to the plasma membrane. Mutant pp6O-src molecules which are not myristylated remain in the cytoplasm and do not transform cells (10, 15).The structural requirements for myristylation of a polypeptide chain have recently been elucidated. Myristate is attached through an amide linkage with a glycine residue at the amino terminus of the acceptor protein (7,24,34) by N-myristyl transferase (NMT). An amino-terminal glycine is essential but not sufficient for myristylation, and a serine or threonine residue is present at position 5 in nearly all myristylated proteins (39, 40). Additional sequence determinants are important, since deletions within the first 10 amino acids of the mature pp6v-vsrc polypeptide sequence abolish myristylation of the resultant protein (26,27). However, no clear consensus sequence seems to exist among all of the known myristylated proteins.Myristylation of both viral and cellular proteins has been shown to be an early event during protein biosynthesis. Treatment of cells with cycloheximide, an inhibitor of protein synthesis, results in immediate cessation of myristylation (5,18,20,22). Moreover, nascent polypeptide chains which are covalently associated with tRNA are already myristylated (44), implying that myristylation occurs cotranslationally. Exactly when t...

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Upon a shift to high temperature, Escherichia coli increase their rate of protein degradation and also the expression of a set of "heat shock" genes. Nonsense mutants of htpR (also called hin), suppressed by a temperature-sensitive suppressor, show lower expression of heat shock genes at 30 degrees C and fail to respond to a shift to 42 degrees C. These mutants were found to have a lower capacity to degrade abnormal or incomplete proteins than that of wild-type cells. This reduction in proteolysis equals or exceeds that in lon mutants, which encode a defective ATP-dependent protease, protease La, and is particularly large in htpR lon double mutants. The activity of protease La was higher in wild-type cells than in htpR mutants grown at 30 degrees C and increased upon shift to 42 degrees C only in the wild type. To determine whether htpR influences transcription of the lon gene, a lon-lacZ operon fusion was utilized. Introduction of the htpR mutation reduced transcription from the lon promoter at 30 degrees C and 37 degrees C. This defect was corrected by a plasmid (pFN97) carrying the wild-type htpR allele. Induction of the heat shock response with ethanol had little or no effect in htpR mutants but stimulated lon transcription 2-3 fold in wild-type cells and htpR cells carrying pFN97. Thus, lon appears to be a heat shock gene, and increased synthesis of protease La under stressful conditions may help to prevent the accumulation of damaged cellular protein.

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Somatic hypermutation and selection of immunoglobulin (Ig) variable (V)-region genes, working in concert, appear to be essential for memory B-cell development in mammals. There has been substantial progress on the nature of the cis-acting DNA elements that regulate hypermutation. The data obtained suggest that the mechanisms of Ig gene hypermutation and transcription are intimately intertwined. While it has long been appreciated that stringent phenotypic selection forces are imposed on the somatically mutated Ig V regions generated during a T-cell dependent B-cell response, the mechanisms involved in this selection have remained enigmatic. Our studies have questioned the role of foreign antigen deposited on follicular dendritic cells in affinity-based positive selection of V regions, and have shown that this selection takes place in a "clone-autonomous" fashion. In addition, our data strongly suggest that affinity for antigen alone is not the driving force for selection of B-cell clones into the memory compartment. In contrast, we suggest that a combination of positive selection for increased foreign antigen binding, and negative selection of antibody V regions that are autoreactive at the onset of the response, or have acquired autoreactivity via hypermutation, results in the "specificity maturation" of the memory B-cell response.