WhiA, a protein of unknown function conserved among gram-positive bacteria, is essential for sporulation in Streptomyces coelicolor A3(2).

The whiA sporulation gene of Streptomyces coelicolor A3(2), which plays a key role in switching aerial hyphae away from continued extension growth and toward sporulation septation, was cloned by complementation of whiA mutants. DNA sequencing of the wild-type allele and five whiA mutations verified that whiA is a gene encoding a protein with homologues in all gram-positive bacteria whose genome sequence is known, whether of high or low G+C content. No function has been attributed to any of these WhiA-like proteins. In most cases, as in S. coelicolor, the whiA-like gene is downstream of other conserved genes in an operon-like cluster. Phenotypic analysis of a constructed disruption mutant confirmed that whiA is essential for sporulation. whiA is transcribed from at least two promoters, the most downstream of which is located within the preceding gene and is strongly up-regulated when colonies are undergoing sporulation. The up-regulation depends on a functional whiA gene, suggesting positive autoregulation, although it is not known whether this is direct or indirect. Unlike the promoters of some other sporulation-regulatory genes, the whiA promoter does not depend on the sporulation-specific sigma factor encoded by whiG.

Duplicated gene clusters suggest an interplay of glycogen and trehalose metabolism during sequential stages of aerial mycelium development in Streptomyces coelicolor A3(2).

DNA sequencing and operon disruption experiments indicate that the genes glgBI and glgBII, which code for the two developmentally specific glycogen branching enzymes of Streptomyces coelicolor A3(2) each form part of larger duplicated operons consisting of at least four genes in the order pep1-treS-pep2-glgB. The sequences of the TreS proteins are 73% identical (93% similar) to that of an enzyme that converts maltose into trehalose in Pinmelobacter, a distantly related actinomycete; and the Pep1 proteins show relatedness to the alpha-amylase superfamily. Disruptions of each operon have spatially specific effects on the nature of glycogen deposits, as assessed by electron microscopy. Upstream of the glgBI operon, and diverging from it, is a gene (glgP) that encodes a protein resembling glycogen phosphorylase from Thermatoga maritima and a homologue in Mycobacterium tuberculosis. These three proteins form a distinctive subgroup compared with glycogen phosphorylases from most other bacteria, which more closely resemble the enzymes from eukaryotes. Diverging from the glgBII operon, and separated from the pep1 gene by two very small ORFs, is a gene (glgX) encoding a probable glycogen debranching enzyme. It is suggested that most of these gene products participate in the developmentally modulated interconversion of immobile, inert glycogen reservoirs, and diffusible forms of carbon, both metabolically active (e.g. glucose-1-phosphate generated by glycogen phosphorylase) and metabolically inert but physiologically significant (trehalose).

A glgC gene essential only for the first of two spatially distinct phases of glycogen synthesis in Streptomyces coelicolor A3(2).

By using a PCR approach based on conserved regions of ADP-glucose pyrophosphorylases, a glgC gene was cloned from Streptomyces coelicolor A3(2). The deduced glgC gene product showed end-to-end relatedness to other bacterial ADP-glucose pyrophosphorylases. The glgC gene is about 1,000 kb from the leftmost chromosome end and is not closely linked to either of the two glgB genes of S. coelicolor, which encode glycogen branching enzymes active in different locations in differentiated colonies. Disruption of glgC eliminated only the first of two temporal peaks of ADP-glucose pyrophosphorylase activity and glycogen accumulation and prevented cytologically observable glycogen accumulation in the substrate mycelium of colonies (phase I), while glycogen deposition in young spore chains (phase II) remained readily detectable. The cloned glgC gene therefore encodes an ADP-glucose pyrophosphorylase essential only for phase I (and it is therefore named glgCI). A second, phase II-specific, glgC gene should also exist in S. coelicolor, though it was not detected by hybridization analysis.

Characterization of spaA, a Streptomyces coelicolor gene homologous to a gene involved in sensing starvation in Escherichia coli.

A Streptomyces coelicolor gene, called spaA, homologous to the stationary phase regulatory gene rspA of Escherichia coli [Huisman and Kolter (1994) Science 265, 537-539], was cloned using the Streptomyces ambofaciens rspA homologue spa2 [Schneider et al. (1993) J. Gen. Microbiol. 139, 2559-2567] as a probe. Considerable differences in sequence and in genetic context were detected between spa2 of S. ambofaciens and spaA of S. coelicolor. A cloned internal fragment of spaA was used to direct integration of a phage vector into the spaA gene. The disruption caused delayed antibiotic production (undecylprodigiosin and actinorhodin) and led on further incubation to increased actinorhodin production at high, but not low, cell density. This phenotype was apparent only on the nutritionally poorest of three media tested. The attempted use of an integrating plasmid-based system for gene replacement of spaA gave rise to extensive deletions of adjacent chromosomal DNA.

Diagnosis and incidence of prion (Creutzfeldt-Jakob) disease: a retrospective archival survey with implications for future research.

Reliable identification of Creutzfeldt-Jakob disease (CJD) in the UX has become essential following the suggestion that prion disease in cattle (BSE) might transmit, accidentally, to humans who eat contaminated beef. Recent data suggest that some cases of CJD may be clinically unrecognized; in order to examine this proposal we reviewed all cases of dementia (n = 1000+) collected in the Runwell Hospital Brain Archive between 1964 and 1990. We identified 19 cases of spongiform encephalopathy of which only 11 were diagnosed before death. These 11 individuals had a characteristic clinical history of CJD (relentless mental deterioration, prominent motor signs and death within a year). Their brains showed little or no external abnormality. In contrast, only two of the eight clinically unrecognized cases had characteristic symptoms. The remaining six presented atypically; their illness lasted 3 years or more, motor signs were much less evident, and simple dementia was the most prominent feature. The brains showed moderate or severe cerebral atrophy. Our data indicate that only about 60% of prion disease cases with pathologically typical spongiform encephalopathy were identified clinically during life. This suggests that human prion disease may be more common than previously supposed and that a further review of the epidemiology of the disease is required.

Tissue-specific glycogen branching isoenzymes in a multicellular prokaryote, Streptomyces coelicolor A3(2).

In the overtly differentiated colonies of Streptomyces coelicolor A3(2), discrete phases of glycogen synthesis are found at the vegetative/aerial mycelium boundary (phase I) and in the immature spore chains at aerial hyphal tips (phase II). We have characterized two S. coelicolor glgB genes encoding glycogen branching enzyme, which are well separated in the genome. Disruption of glgBl led to the formation of abnormal polyglucan deposits at phase I, with phase II remaining normal, whereas disruption of glgBII interfered specifically with phase II deposits, and not with those of phase I. Thus, each branching enzyme isoform is involved in a different phase of glycogen synthesis. This situation contrasts with that in simple bacteria, which typically have a single set of enzymes for glycogen metabolism, and more closely resembles that in plants.

Chromosome 14 linked familial Alzheimer's disease. A clinico-pathological study of a single pedigree.

The clinical features of three affected members of a British pedigree with familial Alzheimer's disease are presented. This pedigree is one of six included in an earlier study which demonstrated linkage to chromosome 14. The individuals were investigated clinically and neuropsychologically, using both PET and MRI over a 4-year period. Further information from three deceased individuals was obtained, including histopathological confirmation of Alzheimer's disease in one case which came to autopsy. The mean age at onset for this family was 43 years. Neurological examination revealed myoclonic jerks in all cases, and one patient was documented to have seizures. Strikingly similar neuropsychological profiles were observed, characterized by an initial memory deficit with early dyscalculia and an impairment in speech production with relative absence of anomia. All individuals showed mild degrees of cerebral atrophy and two individuals had periventricular white matter lesions. PET scanning using [18F]fluorodeoxyglucose showed parieto-temporal hypometabolism in all cases and the two severely affected patients with speech production changes had additional left-sided frontal hypometabolism involving Broca's area. The least affected case initially had a more asymmetrical reduction in metabolism in the left inferior temporal and supramarginal gyri; a follow-up scan showed that this deficit had become bilateral and more severe. These clinical and neuroimaging features have not been previously reported in chromosome 14 linked pedigrees; the phenotypic variability between families suggests allelic heterogeneity at the chromosome 14 locus.

Sequence of the essential early region of phi C31, a temperate phage of Streptomyces spp. with unusual features in its lytic development.

The temperate phage phi C31 is the most studied bacteriophage infecting Streptomyces spp., and has been used to develop an extensive and widely used series of cloning vectors. The sequence of 10 kb of phi C31 DNA containing most or all of the essential early genes was determined. Among the ORFs, 14 (perhaps 15) appear to be protein-coding, and these have been designated ORF1 to ORF14 and ORFX. Previously mapped transcripts appear to initiate upstream from ORFs 1, 8, 11 and 12, and within ORF3 and ORF12, in each case close to one example of the unusual ('21-mer') sequences that appear to serve as a recognition site for RNA polymerase early in the phi C31 lytic cycle [Ingham et al., Mol. Microbiol. 9 (1993) 1267-1274]. Further copies of the 21-mer are upstream from ORF2 and ORF13. There are four recognisable examples of a conserved inverted repeat sequence motif (CIR) thought to bind phi C31 repressor [Smith and Owen, Mol. Microbiol. 5 (1991) 2833-2844]. Only one CIR is closely associated with a 21-mer sequence, though three are located between known transcription units. Of all 14 ORFs, only one (ORF11) would encode a protein unmistakably resembling other known proteins; its product appears to be a DNA polymerase. Strikingly, two codons, TTA (Leu) and AGG (Arg), are absent from the 14 ORFs.

Clinical correlates of postmortem brain changes in schizophrenia: decreased brain weight and length correlate with indices of early impairment.

From a postmortem study of the brains of 56 patients with schizophrenia and 56 controls, 38 cases whose clinical state had been objectively documented in life were examined to determine whether relations existed between features of the illness and postmortem findings. Decreased brain weight was significantly related (p < 0.05) to poor premorbid global function and to poor academic record, and decreased brain length was related to poorer premorbid global function (p < 0.05) and more severe negative symptoms. These relations are consistent with the view that morphological changes in the brain occur early in the course of the disease--that is, they are in some sense "developmental." An excess of "focal damage" in the patient group relative to controls was unrelated to the presence of morphological change or to features of illness, but was more common in female schizophrenic patients and was also correlated with evidence of cerebrovascular disease. This may possibly be due to a discrepancy between the groups in mode and cause of death.

Induction of beta (A4)-amyloid in primates by injection of Alzheimer's disease brain homogenate. Comparison with transmission of spongiform encephalopathy.

Amyloid plaques, associated with argyrophilic dystrophic neurites, and cerebral amyloid angiopathy (CAA), but no neurofibrillary tangles, were found in the brains of three middle-aged marmoset monkeys that had been injected intracerebrally (ic) 6-7 yr earlier with brain tissue from a patient with early-onset Alzheimer's disease. Such changes were not found in the brains of three age-matched control marmosets. Immunochemically the amyloid plaques and CAA stained with antibody to beta (A4)-protein. The plaques and CAA displayed dichroic birefringence when stained with Congo red and viewed under polarized light. beta (A4)-amyloid plaques and CAA were also found in the brain of one of two marmosets injected ic 6 yr previously with brain tissue from a patient with prion disease with concomitant beta (A4)-amyloid plaques and CAA. An occasional beta (A4)-amyloid plaque was found in the brains of two of four marmosets injected ic > 4.5 yr previously with brain tissue from three elderly patients, two of whom had suspected (but untransmitted) CJD. No beta (A4)-amyloid plaques or CAA were found in six marmosets who were older than the injected animals, in four marmosets that had not developed spongiform encephalopathy (SE) having been injected several years previously with human brain tissue from three younger patients with suspected or atypical prion disease, or in 10 younger marmosets who had undergone various neurosurgical procedures. Seventeen marmosets injected in the same way with brain tissue from patients or animals with SE developed SE 17-49 mo after injection. These results suggest that beta (A4)-amyloidosis is a transmissible process comparable to the transmissibility of SE.

Epilepsy, psychosis, and schizophrenia: clinical and neuropathologic correlations.

This study examines the relationship between epilepsy and psychosis. It compares clinical, EEG, and neuropathologic data from a group of subjects who had both epilepsy and psychosis with similar information from another group of patients who had epilepsy but no evidence of psychotic illness. We examined, blind to clinical diagnosis, gross and microscopic material from whole-brain specimens from 10 patients diagnosed with epilepsy plus schizophrenia-like psychosis, nine subjects diagnosed with epilepsy plus "epileptic psychosis," and 36 individuals with epilepsy (21 from an epileptic colony and 15 from the community at large) who had no history of psychosis (n = 10 + 9 + 21 + 15 = 55). We abstracted case histories without knowledge of pathologic findings. Epileptic colony patients had an earlier age at onset of seizures, while epileptic colony and epileptic psychosis patients had more frequent seizures. Epileptic individuals in the community died at a younger age than did epileptic patients in long-stay hospital care. Psychotic epileptic patients had larger cerebral ventricles, excess periventricular gliosis, and more focal cerebral damage compared with epileptic patients who had no psychotic illness. Epileptic patients with schizophrenia-like psychosis were distinguished from all other groups by a significant excess of pinpoint perivascular white-matter softenings. We found that mesial temporal sclerosis and temporal lobe epilepsy occurred with equal frequency in the psychotic and nonpsychotic groups; generalized seizures occurred more frequently in the psychotic epileptics and the epileptic colony epileptics than in the community epileptic controls.

Evidence for the experimental transmission of cerebral beta-amyloidosis to primates.

The brains of three marmosets (Callithrix jacchus) injected intracerebrally 6-7 years earlier with brain tissue from a patient with early onset Alzheimer's disease were found to contain moderate numbers of amyloid plaques with associated argyrophilic dystrophic neurites and cerebral amyloid angiopathy but no neurofibrillary tangles. The plaques and vascular amyloid stained positively with antibodies to beta (A4)-protein. The brains of three age-matched control marmosets from the same colony did not show these neuropathological features. The brain of one of two marmosets injected with brain tissue from a patient with prion disease with concomitant beta-amyloid plaques and cerebral amyloid angiopathy also showed beta-amyloid plaques and angiopathy but no spongiform encephalopathy. An occasional plaque was found in the brains of two of four marmosets injected with brain tissue from three elderly patients with age-related pathology, two of whom had an additional diagnosis of possible prion disease. Neither plaques nor cerebral amyloid angiopathy were found in six other marmosets who were older than the injected animals, in 12 further marmosets who were slightly younger but who had been injected several years previously with brain tissue which did not contain beta-amyloid, or in 10 younger marmosets who had been subjected to various neurosurgical procedures. These results suggest that cerebral beta-amyloidosis may be induced by the introduction of exogenous amyloid beta-protein.

Experimental induction of beta-amyloid plaques and cerebral angiopathy in primates.

Moderate numbers of amyloid plaques with associated argyrophilic dystrophic neurites and cerebral amyloid angiopathy (CAA) but no neurofibrillary tangles (NFTs) were found in the brains of 3 middle-aged common marmosets (Callithrix jacchus) inoculated intracerebrally (i.c.) 6-7 years earlier with brain tissue from a patient with early onset Alzheimer's disease. The plaques and vascular amyloid stained positively with antibodies to beta (A4)-protein. The brains of 3 age-matched control marmosets from the same colony did not show these neuropathological features. beta-amyloid plaques and CAA (but no spongiform encephalopathy) were also found in the brain of a marmoset inoculated with brain tissue from a patient with prion disease with concomitant beta-amyloid plaques and CAA. An occasional beta-amyloid plaque was found in the brains of two marmosets inoculated with brain tissue from elderly patients. No beta-amyloid plaques nor CAA were found in 6 other marmosets who were older than the inoculated marmosets, 10 further marmosets who were slightly younger but who had been inoculated several years previously with brain tissue which did not contain beta-amyloid, and 10 younger marmosets who had been subjected to various neurosurgical procedures. These results suggest that beta-amyloidosis is a transmissible process.

Multiple novel promoters from the early region in the Streptomyces temperate phage phi C31 are activated during lytic development.

Evidence is presented that transcription of most of the early genes in the Streptomyces coelicolor A3(2) phage phi C31 is from a series of unusual promoters that depend on a function expressed early in the phi C31 lytic cycle. Primer extension analysis on the 5' ends of three early mRNAs, from samples prepared 10 min after induction of a thermosensitive phi C31 lysogen, showed that the 5' ends all mapped close to highly similar sequences, which are proposed to be an important part of phage-specific promoters. In a shotgun cloning experiment, a fragment containing one of these sequences strongly activated transcription of the xyIE reporter gene in plaques of a phi C31-derived promoter-probe vector. Another of the sequences was inserted into a xyIE-containing promoter-probe plasmid vector, and promoted xyIE expression only when the host was supporting the lytic cycle of phi C31. This suggested that a transcription factor needed for activity of the promoters was present only in phi C31-infected cells. Examination of published and unpublished phi C31 sequence data revealed several more sequences that closely resemble the conserved region of the characterized promoters. Most of these are found in positions close to apparent transcription start sites mapped previously by low-resolution S1 mapping. An overall consensus sequence for the conserved region suggests a general organization (though not a primary sequence) resembling that of promoters recognized in other bacteria by the sigma 54 form of RNA polymerase.

Ubiquitination as a probe for neurodegeneration in the brain in schizophrenia: the prefrontal cortex.

Abnormalities in brain structure and brain function have been described in schizophrenia. It is not yet known whether these are caused by an abnormality of brain development, some form of birth injury, or a neurodegenerative process. Using immunocytochemical methods and a marker for neurodegeneration (ubiquitin), we examined an area of prefrontal cortex from elderly schizophrenic and control subjects for the presence of ubiquitin-positive degeneration products. There was no statistical difference in the degree of ubiquitination between the control and the patient samples. The findings provide no evidence to support a neurodegenerative process.

Quantitative differences in the deposition of beta A4 protein in the sulci and gyri of frontal and temporal isocortex in Alzheimer's disease.

The distribution of beta-amyloid protein (beta A4) in the frontal and temporal isocortex of 14 Alzheimer's disease brains was examined using a combination of immunohistochemistry and computer image analysis. The area of cortex covered by beta A4 deposits was determined and expressed as a percentage of the total cortical grey matter area in each field of interest. Significantly more beta A4 was found in the grey matter of the sulci as compared to that of the gyral crests in both the frontal and the temporal lobes (P less than 0.05). Furthermore, in each case, greater quantities of beta A4 were observed in the frontal rather than the temporal lobes. This apparent differential vulnerability is likely to reflect underlying anatomical connections or perhaps differences in cell packing density and appears to strengthen the case for an anatomical basis for the spread of the disease pathology.

Phage vectors that allow monitoring of transcription of secondary metabolism genes in Streptomyces.

We describe a bacteriophage phi C31-based system that permits the transcriptional fusion of the convenient reporter gene xylE to chromosomally located promoters in Streptomyces hosts. Applicability of the system to genes for secondary metabolism is demonstrated in an experiment showing that transcription of genes for actinorhodin production in Streptomyces coelicolor A3(2) depends on a transfer RNA gene (bldA) for the rare UUA codon. Two other phi C31::xylE vectors are described that allow detection of promoter activity away from their natural location, either at single copy in a prophage or during lytic infections in plaques.

Discovery of an insertion sequence, IS116, from Streptomyces clavuligerus and its relatedness to other transposable elements from actinomycetes.

We have identified an insertion sequence, IS116, present in Streptomyces clavuligerus at one copy per genome. The element was discovered as a 1.4 kb insertion into the multicopy plasmid pIJ702 after propagation in S. clavuligerus. The nucleotide sequence of IS116 and the flanking sequences from pIJ702 have been determined. The junctions with pIJ702 show no target site duplication and there are no inverted repeats at the ends of the element. One putative coding open reading frame of 1197 bp was identified which would code for a protein product of 399 amino acids. This protein resembles deduced integrase/transposase proteins specified by three other transposable elements of actinomycetes: IS110 and the mini-circle from Streptomyces coelicolor A3(2), and--most particularly--IS900 of Mycobacterium paratuberculosis. Two regions that are relatively conserved among these gene products show features found in similar positions in many reverse transcriptases. IS116 and IS900 are also closely similar in their general organization and (apparently) in their insertion site specificity, whereas IS110 and the mini-circle are quite different in these features.