Loss of adenylyl cyclase I activity disrupts patterning of mouse somatosensory cortex.

The somatosensory (SI) cortex of mice displays a patterned, nonuniform distribution of neurons in layer IV called the 'barrelfield' (ref. 1). Thalamocortical afferents (TCAs) that terminate in layer IV are segregated such that each barrel, a readily visible cylindrical array of neurons surrounding a cell-sparse center, represents a distinct receptive field. TCA arbors are confined to the barrel hollow and synapse on barrel-wall neurons whose dendrites are oriented toward the center of the barrel. Mice homozygous for the barrelless (brl) mutation, which occurred spontaneously in ICR stock at Université de Lausanne (Switzerland), fail to develop this patterned distribution of neurons, but still display normal topological organization of the SI cortex. Despite the absence of barrels and the overlapping zones of TCA arborization, the size of individual whisker representations, as judged by 2-deoxyglucose uptake, is similar to that of wild-type mice. We identified adenylyl cyclase type I (Adcy1) as the gene disrupted in brl mutant mice by fine mapping of proximal chromosome 11, enzyme assay, mutation analysis and examination of mice homozygous for a targeted disruption of Adcy1. These results provide the first evidence for involvement of cAMP signalling pathways in pattern formation of the brain.

Sequence variation in the LEU2 region of the saccharomyces cerevisiae genome.

The LEU2 regions present on yeast plasmid vectors come from two sources, a series of strains derived from S288c and strain M127. The LEU2 region from the S288c series contains a Tyl-17 element with its associated delta sequences and a small repetitive RNA gene while the LEU2 region from M127 which is present on pJDB248, lacks the Tyl-17 element, but carries a delta sequence and a small RNA gene. The various LEU2 plasmids currently in use vary with respect to these sequences depending on which restriction fragment from the region is present on the recombinant molecule. In addition, strain M127 contains three LEU2 homologous sequences that are represented by different EcoRI fragments and which segregate independently at meiosis. Therefore, there are at least four forms of the centromere-distal EcoRI fragment of the LEU2 locus in the Saccharomyces cerevisiae gene pool; these are 7.1 kb, 1.9 kb, 1.48 kb and 1.15 kb long.

Factors affecting heterologous gene expression in Saccharomyces cerevisiae.

The 'promoter' fragment from the yeast phosphoglycerate kinase (PGK) gene has been used to direct the expression of human interferon-alpha-2 (IFN alpha 2) on a high-copy-number plasmid in yeast. The yields of IFN alpha 2 are only 1-3% of yeast total protein, whereas the maximum yield of PGK produced by the PGK gene on a high-copy-number plasmid is at least 50%. IFN alpha 2 is turned over more rapidly than PGK but in addition a major reason for the relatively low level of IFN alpha 2 is that IFN-specific RNA levels are much lower. This does not reflect differences in plasmid copy number or integrity, or differences in the 5' and 3' untranslated regions of the transcripts or DNA flanking regions. It appears that the presence of heterologous coding sequences, or the absence of specific yeast sequences causes a reduction in heterologous RNA levels in yeast.

Efficient synthesis of enzymatically active calf chymosin in Saccharomyces cerevisiae.

We have constructed a high-efficiency expression vector to direct the synthesis of heterologous polypeptides in yeast. The vector is termed a sandwich expression vector as the heterologous gene is inserted between the 5' and 3' control regions of the efficiently expressed yeast PGK gene. We have used this vector to direct the expression of three derivatives of the calf chymosin cDNA gene; preprochymosin, prochymosin and chymosin. Prochymosin is synthesised to at least 5% of total yeast-cell protein and furthermore, it can be readily activated to produce an enzyme which has milk-clotting activity.

Case report: appearance of uterine cervical lymphoma on MRI: a case report and review of the literature.

We present the appearances on CT and MRI of a case of non-Hodgkin's lymphoma (NHL) of uterine cervix. A 41-year-old woman presented with a short history of urinary symptoms and menorrhagia. Previous cervical smears were normal. Clinically, the cervix was replaced by a huge ulcerating mass. Biopsy showed malignant high grade B-cell NHL. T(2) weighted MRI of the pelvis showed a 12 cm intermediate signal mass replacing the cervix, with infiltration of the vagina and left parametrium, and bilateral internal iliac lymphadenopathy. Whole body CT imaging showed lymphoma in the kidneys and pancreas, the latter associated with biliary obstruction. The patient is in complete remission 7 months post chemotherapy, radiotherapy and stenting of biliary stricture. The success of the cervical cancer screening programme has lead to a reduction in the number of cases of advanced cervical carcinoma and the presence of an unusually large homogeneous cervical tumour, with relatively scant necrosis should prompt suspicion of a less common histology such as NHL.

Malaria in England: a geographical and historical perspective.

The marshlands of coastal southern and eastern England had unusually high levels of mortality from the sixteenth to the nineteenth century. The unhealthiness of the environment aroused frequent comment during this period and it was attributed to an endemic disease known as "marsh fever" or "ague". Marsh parishes were perceived both as a danger to the local inhabitants and as a deterrent to potential settlers. This paper traces the geography and history of the "marsh fever" in England and shows that the disease was, in fact, malaria transmitted by anopheline mosquitoes. Malaria, once endemic in the coastal marshes of England, had a striking impact on local patterns of disease and death. Yet this study also suggests that the species of malaria endemic in England were vivax and malariae and not the tropical strains of P. falciparum. The paper outlines a number of ways in which "benign" forms of malaria, acting either directly or indirectly, as well as in conjunction with other factors, could have given rise to the unusually high death rates experienced in early modern marshland England. The discussion concludes with an examination of the reasons for the clinical disappearance of malaria during the nineteenth century, its reappearance after the First and Second World Wars and the problem of imported malaria in Britain today.

Bitter-sweet solutions for malaria: exploring natural remedies from the past.

This paper explores "a wonderful cure" for malaria used successfully by Robert Talbor, an apothecary's apprentice in the English marshes, to treat Essex smugglers and European Royalty in the seventeenth century. The basis of this cure is identified as "quinquina" from the bark of the South American Cinchona tree. The story of Robert Talbor and his secret remedy for malaria opens up a set of intriguing questions about the early history of "quinquina", the subsequent development of quinine, the use of higher plants for antimalarial drugs, including the Chinese plant Artemisia annua L., and the value of unlocking the secrets of the past in our search for strategies to control malaria.

The malariology centenary.

The year 1898 was one of the most significant years in the history of malariology. One hundred years later scientists gathered at the Accademia Nazionale dei Lincei, Rome, to commemorate the Malariology Centenary. This paper provides a short overview of some of the key developments and discoveries in malaria research which took place at the end of the 19th century. The major contributions of Alphonse Laveran, Patrick Manson, Ronald Ross, Battista Grassi and a number of scientists of the Italian School of Malariology to the understanding of the transmission of malaria by Anopheles mosquitoes are described. This paper also highlights the importance of an historical perspective in furthering our understanding of the 'Malaria Challenge after One Hundred Years of Malariology'.

Malaria control in East Africa: the Kampala Conference and the Pare-Taveta Scheme: a meeting of common and high ground.

The 1950 Malaria Conference in Equatorial Africa, held in Kampala, Uganda, has been remembered primarily for its decision to control malaria '...by modern methods as soon as feasible, whatever the original degree of endemicity, and without awaiting the outcome of further experiments.' This decision was far from conclusive and, indeed, reflects only one side of the argument which brought two groups of malariologists into direct opposition on the wisdom of malaria control in equatorial Africa, using modern methods such as DDT. Through an examination of the unpublished verbatim transcript of the Kampala Conference, we are able to document the 'furious debates' which took place at Kampala in 1950. We highlight, in particular, the adamant concerns expressed by some of the delegates that intervention in areas of high malaria transmission might lead to a loss of naturally acquired immunity which, in turn, could give rise to a resurgence of malaria, should the control strategies fail to be sustained. As we show, this concern had been expressed by a number of malariologists working in East Africa in the first half of the twentieth century, but it was only with the advent of DDT, as a residual insecticide, that the implications of wide-spread control, in the absence of any knowledge of the long-term consequences, became a serious possibility. While the Kampala Conference gave the 'go ahead' to control malaria in Africa without awaiting the outcome of 'further experiments', a number of participants insisted that a field trial should be set up to evaluate the impact of malaria on areas of high transmission both before and after spraying: to this end, a field trial in Pare-Taveta was carried out in 1954-59. In this paper we look at the Kampala Conference for its scientific debates and the Pare-Taveta Scheme for its field applications. In the final part of the paper, we address a number of questions raised at Kampala which have, once more, become contentious issues, following the recent successful trials of ITBNs. We believe that an understanding of the historical foundations of these issues should provide an important component of the new WHO campaign to Roll Back Malaria.

Regulation of isopenicillin N synthetase (IPNS) gene expression in Acremonium chrysogenum.

Total RNA was extracted daily from the beta-lactam antibiotic producing fungus A. chrysogenum strain CO728 during a 7 day cephalosporin C fermentation. IPNS mRNA species, with a size of about 1.5 kb, were detected by Northern blotting at high levels between days 2 and 4. The rapid appearance of IPNS mRNA in mycelial extracts up to day 2 suggests that IPNS is regulated at the transcriptional level. Primer extension and S1 endonuclease mapping studies indicate the existence of two major and at least two minor transcription initiation start sites. There was no change in the relative levels of the four transcripts during the period they could be detected. A region upstream of the IPNS structural gene (pcbC) has been sequenced and the transcription initiation sites appear as major and minor pairs on either side of one of the pyrimidine-rich blocks that punctuate the promoter sequence.

Stabilization of yeast artificial chromosome clones in a rad54-3 recombination-deficient host strain.

The cloning and propagation of large fragments of DNA on yeast artificial chromosomes (YACs) has become a routine and valuable technique in genome analysis. Unfortunately, many YAC clones have been found to undergo rearrangements or deletions during the cloning process. The frequency of transformation-associated alterations and mitotic instability can be reduced in a homologous recombination-deficient yeast host strain such as a rad52 mutant. RAD52 is one member of an epistatic group of genes required for the recombinational repair of double-strand breaks in DNA. rad52 mutants grow more slowly and transform less efficiently than RAD + strains and are therefore not ideal hosts for YAC library construction. We have investigated the ability of both null and temperature-sensitive alleles of RAD54 , another member of the RAD52 epistasis group, to prevent rearrangements of human YAC clones containing tandemly repeated DNA sequences. Our results show that the temperature-sensitive rad54-3 allele blocks mitotic recombination between tandemly repeated DYZ3 satellite sequences and significantly stabilizes a human DYZ5 satellite-containing YAC clone. Yeast carrying the rad54-3 mutation can undergo meiosis, have growth and transformation rates comparable with RAD + strains, and therefore represent improved YAC cloning hosts.

Loss of 2 um DNA from Saccharomyces cerevisiae transformed with the chimaeric plasmid pJDB219.

Two plasmids containing Saccharomyces cerevisiae 2 µm DNA sequences and the S. cerevisiae LEU2 gene have been found to display incompatibility with 2 µm DNA; in the presence of the LEU2 plasmids, 2 µm DNA can be lost. The LEU2 plasmids can be lost spontaneously after (and before) 2 µm DNA loss has occurred, so that strains completely lacking 2 µm DNA sequences can be obtained routinely.

Control of recombination within and between DNA plasmids of Saccharomyces cerevisiae.

[2 µm(+) and [2µm°] yeast were transformed to stable leucine prototrophy with the hybrid yeast - E. coli plasmid, pJDB219. This plasmid contains the entire sequence of the endogenous 2 µm yeast DNA plasmid in addition to the yeast nuclear LEU2 (+) gene and the Co1E1 derivative, pMB9. In the [2 µm(+)] transformants, a new wholly yeast LEU2 (+) plasmid, pYX, was generated, probably by a recombination event between pJDB219 and 2 µm DNA. The plamid, pYX, in the absence of 2 µm DNA, was found to exist in equimolar amounts of two forms, A and B, which probably arise by intramolecular recombination across the inverted repeat sequences of the 2 µm DNA portion of the plasmid. pJDB219 was found to require the presence of 2 µm DNA to undergo this intramolecular recombination. The results suggest that 2, µm DNA and pYX code for a gene product required in this recombination event which pJDB219 cannot produce.

Problem in diagnostic imaging: have you got the nerve?

This article poses a diagnostic problem commonly encountered in neuroradiology and otolaryngology. The solution and ensuing discussion focus on the anatomy of the posterior cranial fossa (with emphasis on the cerebellopontine angle) and the relevant pathology. Current methods of imaging the posterior cranial fossa are explained and their relative merits discussed.