A polybasic domain allows nonprenylated Ras proteins to function in Saccharomyces cerevisiae.

Ras proteins undergo a series of posttranslational modifications prior to association with the cytoplasmic surface of the plasma membrane. The modification steps include farnesylation, proteolysis, methylesterification, and palmitoylation. A 4-amino acid residue motif known as the CaaX box (C is cysteine, a is generally aliphatic, and X is the carboxyl-terminal residue) is the sequence recognized by the prenyl transferase that initiates the modification pathway. As part of our studies to define the requirements for Ras membrane association, we directed mutagenesis to the yeast Ras2 protein CaaX box to assess the relative importance of prenylation, palmitoylation, and stretches of basic amino acids on the function of the protein. The wild type yeast Ras2 protein terminates in the sequence Cys-Cys-Ile-Ile-Ser. We have identified mutations that do not contain a CaaX box but still encode functional Ras proteins. These mutations replace the terminal serine of the CaaX box with the sequence -Lys-Leu-Ile-Lys-Arg-Lys. Three mutants have been analyzed in detail. Ras2(CCIIKLIKRK) functions at a level similar to wild type Ras2, whereas cells expressing only Ras2(SCIIKLIKRK) and Ras2(SSIIKLIKRK) forms of Ras2 protein grow more slowly at 30 degrees C. In addition, strains expressing only Ras2(SSIIKLIKRK) protein fail to grow at 37 degrees C. Replacement of the basic residues with neutral amino acids (Ras2(CCIISIIS)) completely abolishes their ability to support Ras-dependent growth. The extension mutants are not prenylated, but Ras2(CCIIKLIKRK) and Ras2(SCIIKLIKRK) are palmitoylated. These results demonstrate that a diverse set of carboxyl-terminal sequence motifs and posttranslational modifications lead to functional Ras proteins in yeast.

Vectors for the inducible overexpression of glutathione S-transferase fusion proteins in yeast.

A rapid and convenient method of protein purification involves creating a fusion protein with glutathione S-transferase (GST) (Smith and Johnson, Gene 67, 31-40, 1988). In this report, we describe two vectors for the conditional expression of GST fusions in Saccharomyces cerevisiae. The parent plasmid is based on a high-copy, galactose-inducible shuttle vector previously described (Baldari et al., EMBO J. 6, 229-243, 1987). We have demonstrated the use of this system by creating fusions between GST and the yeast RAS2 gene. GST-Ras2 fusion proteins undergo the post-translational modifications required for Ras2p to become membrane localized. These vectors provide a useful system for the expression and purification of eukaryotic proteins requiring post-translational modification.

Normal mitochondrial structure and genome maintenance in yeast requires the dynamin-like product of the MGM1 gene.

The isolation and characterization of MGM1, an yeast gene with homology to members of the dynamin gene family, is described. The MGM1 gene is located on the right arm of chromosome XV between STE4 and PTP2. Sequence analysis revealed a single open reading frame of 902 residues capable of encoding a protein with an approximate molecular mass of 101 kDa. Loss of MGM1 resulted in slow growth on rich medium, failure to grow on non-fermentable carbon sources, and loss of mitochondrial DNA. The mitochondria also appeared abnormal when visualized with an antibody to a mitochondrial-matrix marker. MGM1 encodes a dynamin-like protein involved in the propagation of functional mitochondria in yeast.

Drosophila RNA polymerase II elongation factor DmS-II has homology to mouse S-II and sequence similarity to yeast PPR2.

DmSII is a Drosophila RNA polymerase II elongation factor which suppresses pausing by RNA polymerase II at specific sites on double stranded templates. Using antibodies produced against the purified protein, a Drosophila cDNA expression library was screened and a cDNA was isolated which encoded a portion of DmSII. When this cDNA was used to probe Kc cell mRNA the predominant species was found to be 1.4 kb in length. The original cDNA was used to screen a Drosophila Kc cell cDNA library resulting in the isolation of a 1.4 kb cDNA which was then sequenced. The deduced protein sequence for DmSII exhibited high similarity to mouse SII protein sequence. In addition, significant sequence similarity was found with the protein encoded by the yeast gene PPR2, which is involved in regulation of URA4 gene expression. The comparison of amino acid sequences suggests that DmSII is comprised of two domains homologous to mouse SII separated by a flexible, serine rich region of low homology. The shorter yeast protein has sequence similarity only to the carboxy terminal domain.

Forensic aspects of terrorism.

Terrorist violence is criminal and requires an expert forensic investigation. However, it has little in common with assault and murder in domestic circumstances, the kind of violence experienced by most forensic practitioners. The terrorist activity in Northern Ireland has revealed the differences. Terrorist shootings involve high velocity and automatic weapons and also include cold-blooded executions. Terrorist explosions cause specific injuries which enable the events to be reconstructed. In both shooting and bombing, the investigators have to contend with rumours and allegations which inflame feelings in the locality. All these aspects are described and illustrated. Conclusions are reached that when terrorist violence erupts there must be a forensic service able to cope and that high standards of scientific investigation must always be maintained.

Forensic aspects of terrorism

Terrorist violence is criminal and requires an expert forensic investigation. However, it has little in common with assault and murder in domestic circumstances, The kind of violence experienced by most forensic practitioners. The terrorist activity in northern ireland has revealed the differences. Terrorist shootings involve high velocity and automatic weapons and also include cold-blooded executions. Terrorist explosions cause specific injuries which enable the events to be reconstructed. In both shooting and bombing the investigators have to contend with rumours and allegations which inflame feelings in the locality. All these aspects are described and illustrated. Conclusions are reached that when terrorist violence erupts there must be a forensic service able to cope and that high standards of scientific investigation must always be maintained (AU)

De subitaneis mortibus. XXIX. Randomly distributed focal myocardial lesions causing destruction in the His bundle or a narrow-origin left bundle branch.

Most focally destructive lesions of the heart (e.g., myocarditis) have an essentially random distribution. Any mechanical significance of such diseases depends primarily on there being a very large number of foci. However, single even very small lesions can have profound electrophysiological significance if appropriately located within the cardiac conduction system. Two cases of sudden unexpected death are here reported which had destructive lesions involving the His bundle and a narrow-origin left bundle branch. Not much is known of the electrical or mechanical effects of acute left branch block which is due to complete anatomical transection of that structure in the human heart, and some possible consequences (including sudden unexpected death) are considered. Myocarditis or any other focally destructive disease in the heart may be of little functional importance if it is not widespread, unless some of the lesions happen to damage any of several small but crucially important sites in the cardiac conduction system. Studies to determine the presence or absence of this precise type of damage should be done especially in cases of sudden unexpected death.

De subitaneis mortibus. XX. Cardiac electrical instability in the presence of a left superior vena cava.

Abnormalities of cardiac rhythm have been reported by others in patients with a persistent left superior vena cava. We present the histological findings from postmortem examination of the cardiac conduction system of two such patients. The first patient was a schoolboy who died suddenly and unexpectedly. His sinus node was abnormally small, his A-V node contained numerous venous lacunae and was stretched out beneath the enlarged coronary sinus, both A-V node and His bundle were dispersed in the central fibrous body in a fetal pattern, and isolated fragments of A-V nodal tissue were connected to the myocardium at the crest of the interventricular septum. The second patient complained of palpitations many months prior to surgical correction of an interventricular septal defect; his postoperative course included multiple arrhythmias and he died suddenly on the 16th postoperative day. Although his sinus node was histologically normal, the nutrient artery contained a polypoid fibromuscular mass virtually occluding its lumen; his A-V node and His bundle were also dispersed in the central fibrous body in the fetal pattern, and the A-V node contained numerous venous lacunae as well as being histologically disorganized in its cytological pattern. These anatomical findings may indicate a basis for various forms of cardiac electrical instability, and deserve consideration in the future evaluation of any patient found to have a persistent left superior vene cava, but particularly if there is clinical evidence suggesting an arrhythmia or conduction disturbance.

XVIII. Persistent fetal dispersion of the atrioventricular node and His bundle within the central fibrous body.

In the hearts of two victims of sudden unexpected death (one age 20 years and the other 11 years) there was abnormally delayed persistence of the pattern of fetal dispersion of the A-V (atrioventricular) node and His bundle within the central fibrous body. This pattern is characterized by a splayed or excessively fragmented histological appearance of these structures viewed in cross-section. Some of the fronds of A-V nodal tissue formed loops connecting one portion of the node to another. Other fragments of nodal tissue appeared isolated within the central fibrous body, occasionally connecting directly to the crest of the interventricular septum. A number of the A-V nodal fragments were undergoing resorptive degeneration. Directly adjacent to the A-V node of the 11-year-old subject there was an island of cartilage within the central fibrous body. Since these anatomically separated fragments of A-V nodal tissue were so numerous and varied widely in size and thickness, in length, in histological organization, and in their apparent state of preservation probably extant during life, it is suggested that they form potential routes for abnormal conduction or impulse formation within the A-V junctional region and that some of these electrophysiological disturbances might be quickly lethal.

De subitaneis mortibus. XVII. Multifocal stenoses due to fibromuscular dysplasia of the sinus node artery.

The sinus node artery was focally narrowed by fibromuscular dysplasia in two examples of sudden unexpected death reported here. Although both cases had additional histological abnormalities in the conduction system of the heart, the more striking feature was the focal fibromuscular dysplasia. These findings are discussed in relationship to a large number of similar examples of focal fibromuscular dysplastic narrowing of the sinus node artery observed in other victims of sudden unexpected death, considering some possible mechanisms for lethal electrical instability of the heart and also the possible pathogenesis of such fibromuscular dysplasia.

De subitaneis mortibus. XII. Asymmetrical hypertrophy of the heart.

Subjects with asymmetrical hypertrophy of the heart are prone to sudden death. Neither the pathogenesis of the eccentric hypertrophy nor the mechanism of sudden death is fully understood. In this report we describe certain postmortem findings in the hearts of 22 subjects who died suddenly, silently and unexpectedly, and in whom the only significant abnormality at autopsy was asymmetrical hypertrophy of the heart. Deep clefts were present in the septum in seven hearts, the small coronary arteries were abnormally narrowed in ten, the sinus node was sclerosed by fibrosis in 12, there was variable narrowing of the atrioventricular (A-V) node artery in many and the His bundle was too thin in three. There were multiple cysts or channels in the central fibrous body and of the adjacent A-V node and His bundle in four hearts. Most of the hearts displayed a fetal dispersion of the A-V node and His bundle throughout the central fibrous body, but this was particularly conspicuous in 13 hearts. These abnormalities in all parts of the conduction system suggest a variety of possible mechanisms by which the heart could become electrically unstable but do not indicate that one single mechanism is at fault in all. They offer some explanation for the reported high incidence of atrial fibrillation in such patients, and why they fare so badly with this arrhythmia. While the pathogenesis of asymmetrical hypertrophy may in some part be attributable to narrowed small coronary arteries or to an abnormal sequence or speed of septal and ventricular activation or to mechanical deficiency caused by deep septal clefts, none of these features was universally present in our series. Both asymmetrical hypertrophy of the heart and the sudden death which so frequently accompanies it probably develop by a variety of pathogenetic mechanisms.