Schizophrenia as an anomaly of development of cerebral asymmetry. A postmortem study and a proposal concerning the genetic basis of the disease.

Schizophrenia is associated with structural changes (eg, a mild degree of ventricular enlargement) in the brain, although whether these precede onset of illness or progress with episodes is not established. In a postmortem study, we find that ventricular enlargement affects the posterior and particularly the temporal horn of the lateral cerebral ventricle. By comparison with controls and with patients suffering from Alzheimer-type dementia (in which there is also temporal horn enlargement), the change is highly significantly selective to the left hemisphere. This deviation was not accompanied by an increase in glial cell number (examined chemically by assay of diazepam-binding inhibitor immunoreactivity and microscopically by density of staining with the Holzer technique). The findings are consistent with the view that schizophrenia is a disorder of the genetic mechanisms that control the development of cerebral asymmetry.

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.

Mutational cloning in Streptomyces and the isolation of antibiotic production genes.

An attachment site-deleted derivative, phi C31KC400, of the Streptomyces temperate phage phi C31 was used to clone fragments of the genetic determinants (mmy) for the biosynthesis of an antibiotic, methylenomycin A. The vector carries a cloned viomycin resistance gene (vph), and can transduce a recipient to viomycin resistance when DNA sequences are common to the phage and the recipient: the phage integrates into the recipient's genome through a Campbell type of recombination at the site of the homology. For the cloning of mmy DNA, the homology was provided by the in vitro insertion into the vector of DNA from a methylenomycin A-producing streptomycete. Clones carrying mmy DNA could integrate into a methylenomycin-producing recipient's mmy genes, sometimes disrupting their expression: thus a search of viomycin-resistant transductants for methylenomycin non-producing derivatives identified lysogens which spontaneously released phi C31 phages carrying mmy DNA. Some of these lysogens participated in methylenomycin co-synthesis with previously isolated mmy mutants. At least 7 kb of mmy DNA was identified among the clones. Screening for mmy non-producers was simplified by exploiting the presence of the mmy genes on the (albeit unisolable) plasmid, SCP1. In the course of the experiment, SCP1, a low copy number plasmid in its primary host S. coelicolor A3(2), was shown to have a copy number of about 30 in the single S. parvulus SCP1+ transconjugant strain tested, and a molecular size probably greater than 200 kb.

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.

Restriction mapping of the DNA of the Streptomyces temperate phage phi C31 and its derivatives.

DNA of phi C31 propagated on Streptomyces lividans 66 contained no sites for the restriction enzymes BamHI, SalPI (=PstI) and XhoI; one for XbaI; three for HpaI; five for ClaI and KpnI; six for EcoRI; about 13 for HindIII; about 14 for BclI; and more than 15 for FspAI, HgiAI, SacI, SalGI and SmaI. A complete map of 20 sites (XbaI, HapI, ClaI, KpnI and EcoRI) was obtained using partial digestion and double digestion of DNA of the wild-type and deletion and insertion mutants. The total molecular size was estimated to be 41.2 kb.

Dispensable sequences and packaging constraints of DNA from the Streptomyces temperate phage phi C31.

Deletion mutants of the temperate Streptomyces phage phi C31 were selected by two methods: resistance to the chelating agent sodium pyrophosphate, and plating of a phi C31::pBR322 hybrid phage on Streptomyces albus G to obtain large plaque mutants. The deletions defined a 7.7 kilobase (kb) segment of the phi C31 genome which is inessential for plaque formation, in addition to a shorter segment including the repressor gene. Analysis of deletions and insertions suggested that the minimum size of the phi C31 genome allowing plaque formation is 37.5 kb (91% of the wild-type length of 41.2 kb), and the maximum is at least 42.4 kb (103%). These results indicate that it should be possible to introduce up to 10 kb of foreign DNA into a suitably developed phi C31 vector.

New derivatives of the Streptomyces temperate phage phi C31 useful for the cloning and functional analysis of Streptomyces DNA.

The thiostrepton resistance gene (tsr) of Streptomyces azureus, and a synthetic oligonucleotide adapter sequence, were introduced into the DNA of attP-site-deleted phage phi C31-based cloning vectors. The DNA of two of the new derivatives, KC515 and KC516, contains single sites for the enzymes BamHI, BglII, PstI, PvuII, SstI (two sites close together) and XhoI, available for the insertion of DNA of up to 4 kb. The two vectors also contain a cloned, promoterless viomycin phosphotransferase gene (vph) from Streptomyces vinaceus. When an internal segment of the Streptomyces coelicolor glycerol (gyl) operon was inserted at the appropriate position and in the correct orientation next to vph, it could bring about in vivo recombination leading to fusion of vph of the chromosomally located gyl operon, resulting in glycerol-regulated expression of viomycin resistance. Two other new phi C31 derivatives, KC505 and KC518, are PstI and BamHI replacement vectors, respectively, for 2-8-kb DNA fragments, and allow simple screening for the presence of inserted DNA.

The complete nucleotide sequence of the Pseudomonas gene coding for carboxypeptidase G2.

The complete nucleotide sequence of the Pseudomonas chromosomal gene coding for the enzyme carboxypeptidase G2 (CPG2) has been determined. The nucleotide sequence obtained has been confirmed by comparing the predicted amino acid sequence with that of randomly derived peptide fragments and by N-terminal sequencing of the purified protein. The gene has been shown to code for a 22 amino acid signal peptide at its N-terminus which closely resembles the signal peptides of other secreted proteins. An alternative 36 amino acid signal peptide which may function in Pseudomonas has also been identified. The codon utilisation of the gene is influenced by the high G + C (67.2%) content of the DNA and exhibits a 92.8% preference for codons ending in G or C. This unusual codon preference may contribute to the generally observed weak expression of Pseudomonas genes in Escherichia coli. A region of DNA upstream of the structural gene has also been sequenced and a ribosome binding site and two putative promoter sequences identified.

The expression of Streptomyces and Escherichia coli drug-resistance determinants cloned into the Streptomyces phage phi C31.

Lysogens obtained by infecting Streptomyces albus G with a phi C31-pBR322 chimaeric prophage or its delta W12 deletion derivative had increased tetracycline resistance. The ability of the delta W12 derivative to transduce tetracycline resistance was inactivated by inserting a viomycin resistance determinant (vph) into the BamHI site of the pBR322 tet gene, and restored by excising the vph gene. Another deletion mutant (delta W17) of the chimaera, carrying an intact tet gene, was normally unable to transduce tetracycline resistance. This inability was correlated with the finding, by Southern hybridisation analysis, that the att site required for insertion of phi C31 prophage into the host chromosome was located within the delta W17 deletion. Use of phi C31 lysogenic recipient permitted the integration of the att-deleted phage, presumably by homologous recombination, giving tetracycline-resistant double lysogens. This technique was extended to S. coelicolor A3(2) in the detection of derivatives of the att-deleted phage into which a thiostrepton-resistance determinant (tsr) had been inserted in vitro. Phage released from double lysogens were mainly recombinants. One such recombinant is a PstI vector for DNA cloning, able to accommodate up to 6 kb of introduced DNA.

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.

The restriction mapping of c gene deletions in Streptomyces bacteriophage phi C31 and their use in cloning vector development.

In addition to 20 previously mapped restriction sites in the DNA of phi C31, we have determined eight sites for SphI, four for EcoRV, and two for SstII; there are none for BglII or SstI. Nine sites were in a 12-kb segment of DNA containing no previously mapped sites. Deletions causing clear-plaque morphology were located in this part of the DNA, in a 3-kb interval between an EcoRV and an SphI site at the centre of the DNA molecule. One of the deletions (delta C3) was obtained in a previously described phi C31c+::vph (viomycin phosphotransferase) derivative containing two PstI sites separated by 3.9-kb of inessential DNA. After in vitro PstI treatment, plaque-forming phages lacking the 3.9-kb fragment were obtained from the c+ phage but not from its delta C3 derivative. Thus a 36.2-kb genome, but not one of 34.4 kb, was able to give infectious virions. PstI-generated DNA fragments of up to 8 kb can be inserted in vitro into the delta C3 derivative with retention of the vph selective marker. With the insertion of a 6.03-kb PstI fragment of plasmid SCP2, the latter phage became a potential vector (with loss of vph) for BamHI-generated DNA fragments of up to 9 kb. In the course of this work, several ClaI sites in phi C31::pBR322 bifunctional replicons were shown to be lost when the DNA was propagated in a dam+ Escherichia coli strain. This will allow the use of such replicons for the cloning of ClaI-generated DNA fragments of up to 6.7 kb.

The tyrosyl-tRNA synthetase from Escherichia coli. Complete nucleotide sequence of the structural gene.

The structural component of the tyrS gene of Escherichia coli, comprising 1269 base pairs, has been fully sequenced by the combined M13/dideoxychain termination approach. The gene has a codon usage pattern which is typical of highly expressed proteins and similar to other Escherichia coli aminoacyl-tRNA synthetase genes. Peptide purification and sequencing has been used to locate the N-terminus and to provide confirmation of 95% of the translated protein sequence. This latter yields on Mr of 47,403 for the Escherichia coli tyrosyl-tRNA synthetase, and reveals considerable homology with the primary structure of the analogous enzyme isolated from Bacillus staerothermophilus.

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.

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.

Brain amino acid concentrations and Ca2+-dependent release in intractable depression assessed antemortem.

The concentrations of 3 putative neurotransmitters (glutamate, aspartate and gamma-aminobutyrate), 4 related amino acids and 5 non-transmitter-related amino acids have been measured in neurosurgical samples (frontal cortex) from patients with intractable depression and controls. In addition, the glutamate receptor agonist 2-amino-4-sulpho-butanoic acid (homocysteic acid) has been identified in human brain and measured in these samples. There were no changes in the concentrations of amino acids in depressed patients compared to control with the exception of aspartic and homocysteic acids which were elevated in a sub-group of patients with depression compared to control. The Ca2+-dependent release (K+-stimulated) of putative neurotransmitters has been demonstrated for the first time from brain tissue of depressed patients. Glutamate release was unaltered from the control value. Aspartate values showed unexplained variability but it's release and that of gamma-aminobutyrate were elevated in some depressed subjects. These results do not support the hypothesis of reduced amino acid function in depressive illness.

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.

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.

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.