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PubMed1977

Uptake of horseradish peroxidase by geniculo-cortical axons in the golden hamster: analysis by computer reconstruction.

Micro-injections of horseradish peroxidase (HRP) were made into the visual cortex of the golden hamster. The "projection lines" of labelled neurons in the dorsal lateral geniculate nucleus (LGNd) were three-dimensionally reconstructed, using a computer graphics technique. The lines run rostrally and medially from their origins at the lateral surface of the nucleus. Using an anatomically determined retinotopic map of the LGNd, the positions of all labelled cells near the lateral surface were converted into equivalent visual field co-ordinates and displayed on a physiologically determined retinotopic map of the primary visual cortex. Comparison between the scatter of these equivalent retinotopic loci and an actual reconstruction of the injection site revealed that: 1. there was general agreement between the independent retinotopic maps of LGNd and visual cortex; 2. there was greater retinotopic scatter of labelled LGNd cells than could be accounted for by the area of tissue injury in the cortex; 3. the retinotopic scatter matched more closely the total visible halo of HRP staining in the grey matter; 4. HRP can be taken up from a cytoarchitectonic field into which it diffuses after injection into a neighbouring area; 5. HRP is probably not taken up by undamaged axons in the white matter. These results are compared with those obtained in other animals and other systems. No general rules emerge, but the possibility of uptake from wide areas of diffusion must be considered when interpreting results of HRP injection.

Animals↗
PubMed1989

Isolation and characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by the mating hormone a-factor.

Nine independent mutants which are supersensitive (ssl-) to G1 arrest by the mating hormone a-factor were isolated by screening mutagenized Saccharomyces cerevisiae MAT alpha cells on solid medium for increased growth inhibition with a-factor. These mutants carried lesions in two complementation groups, ssl1 and ssl2. Mutations at the ssl1 locus were mating type specific: MAT alpha ssl1- cells were supersensitive to a-factor but MATa ssl1- were not supersensitive to alpha-factor. In contrast, mutations at the ssl2 locus conferred supersensitivity to the mating hormone of the opposite mating type on both MAT alpha and MATa cells. The alpha-cell specific capacity to inactivate externally added a-factor was shown to be lacking in MAT alpha ssl1- mutants whereas MAT alpha ssl2- cells were able to inactivate a-factor. Complementation analysis showed that ssl2 and sst2, a mutation originally isolated as conferring supersensitivity to alpha-factor to MATa cells, are lesions in the same gene. The ssl1 gene was mapped 30.5 centiMorgans distal to ilv5 on chromosome XII.

Alleles↗
PubMed1991

Human acetylcholinesterase and butyrylcholinesterase are encoded by two distinct genes.

1. Various hybridization approaches were employed to investigate structural and chromosomal interrelationships between the human cholinesterase genes CHE and ACHE encoding the polymorphic, closely related, and coordinately regulated enzymes having butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) activities. 2. Homologous cosmid recombination with a 190-base pair 5' fragment from BuChEcDNA resulted in the isolation of four overlapping cosmid clones, apparently derived from a single gene with several introns. The Cosmid CHEDNA included a 700-base pair fragment known to be expressed at the 3' end of BuChEcDNA from nervous system tumors and which has been mapped by in situ hybridization to the unique 3q26-ter position. In contrast, cosmid CHEDNA did not hybridize with full-length AChEcDNA, proving that the complete CHE gene does not include AChE-encoding sequences either in exons or in its introns. 3. The chromosomal origin of BuChE-encoding sequences was further examined by two unrelated gene mapping approaches. Filter hybridization with DNA from human/hamster hybrid cell lines revealed BuChEcDNA-hybridizing sequences only in cell lines including human chromosome 3. However, three BuChEcDNA-homologous sequences were observed at chromosomal positions 3q21, 3q26-ter, and 16q21 by a highly stringent in situ hybridization protocol, including washes at high temperature and low salt. 4. These findings stress the selectivity of cosmid recombination and chromosome blots, raise the possibility of individual differences in BuChEcDNA-hybridizing sequences, and present an example for a family highly similar proteins encoded by distinct, nonhomologous genes.

Acetylcholinesterase↗
PubMed1984

Further evidence from tryptic peptide analysis for heterogeneity among type D retroviruses.

Tryptic peptide analyses were performed on the major internal structural proteins of type D retroviruses isolated originally from subhuman primate species (MPMV, SMRV, LV) and from permanent human cell lines (HeLaV, HEp-2V, PMFV). The p25 peptide maps of MPMV, LV, PMFV and HEp-2V were very similar but showed a striking dissimilarity to the p25 map of HeLaV and the p35 map of SMRV. All type D viruses included in this study could be distinguished by peptide maps of at least two of their three low-molecular-weight proteins p10, p12 and p15. These studies further demonstrate the heterogeneity among type D retroviruses.

Cell Line↗
PubMed2001

Migration and differentiation of Nkx-2.2+ oligodendrocyte progenitors in embryonic chicken retina.

During early neural development, the Nkx-2.2 homeobox gene is initially induced in the ventral ventricular zone of the central nervous system (CNS). At later stages, Nkx-2.2+ cells migrate away into the surrounding regions and co-express several oligodendrocyte marker genes. Here we report that Nkx-2.2+ cells also migrate into the avian retina from the optic nerve. These Nkx-2.2+ cells are specifically labeled by the early oligodendrocyte markers PDGFR alpha, O4 and GalC, but not the microglial marker QH1 or neuron-specific marker MAP-2. These observations provide further evidence that Nkx-2.2 is selectively expressed in migratory oligodendrocyte progenitor cells and that retina oligodendrocytes originate from the ventral diencephalon.

Animals↗
PubMed2002

Fine mapping of the locus for Shwachman-Diamond syndrome at 7q11, identification of shared disease haplotypes, and exclusion of TPST1 as a candidate gene.

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterised by exocrine pancreatic dysfunction, haematological and skeletal abnormalities. We have previously defined the SDS locus as a 2.7 cM interval spanning the centromere of chromosome 7. To facilitate additional analysis of this complex and poorly characterised region, a framework of ordered genetic markers at 7p11-q11, including six newly identified, has been constructed using somatic cell, radiation hybrid and STS-content mapping. We have identified shared disease haplotypes, that recur in unrelated families of common ethnic origin, and extend across the SDS locus. Detection of ancestral and intrafamilial recombination events in patients refined the SDS locus to a 1.9 cM interval at 7q11, which contains the tyrosylprotein sulfotransferase 1 (TPST1) gene. Patients with SDS were screened for mutations in TPST1 by sequencing of exons and intron-exon junctions. Two single nucleotide polymorphisms, but no disease-causing mutations, were identified. In addition, Southern blot analysis yielded no evidence of large-scale mutations, and RT-PCR analysis failed to detect alterations in expression. These results exclude TPST1 as the causative gene for SDS. The established map of the refined SDS locus will assist in the identification and characterisation of other candidate genes for SDS.

Chromosomes, Human, Pair 7↗
PubMed1986

DNA replication is required for abundant expression of a plasmid-borne late US11 gene of herpes simplex virus type 1.

During herpes simplex virus type 1 (HSV-1) infection, the appearance of true-late gene products is severely reduced under conditions of DNA synthesis inhibition. This report describes the use of a plasmid-borne promoter of a true-late HSV-1 gene (US11), linked to the rabbit beta-globin gene, to study the requirement of DNA replication for late gene expression. The activity of the plasmid-borne US11 promoter in constructs containing or lacking an HSV-1 origin of replication (ORIS) was analysed by quantitative S1 mapping of correctly initiated hybrid transcripts. Following HSV-1 superinfection of transfected HeLa cells, the US11 promoter in ORI+ plasmids was expressed with similar kinetics to the viral US11 promoter. US11 promoter activity was first detected at the same time as the onset of DNA template replication. Expression of US11 RNA was detectable from non-replicating ORI- plasmids, although transcript accumulation was reduced by greater than 90%. Sequences containing the IE-5 promoter (a 3' co-terminal gene whose transcription starts 5' of US11) also played a positive role in achieving normal US11 gene expression.

Cloning, Molecular↗
PubMed1984

Transcription of the mouse ribosomal spacer region.

This paper describes experiments designed to test the hypothesis that DNA sequences upstream from the mouse rRNA promoter are transcribed in vivo or in vitro. Plasmid pB28 contains a SalI restriction fragment that extends from -169 to -1,894 base pairs, with respect to the origin of transcription of pre-rRNA. Labeled RNA synthesized in intact cells does not hybridize to this region. Neither S1 nuclease mapping nor RNA dot blot hybridization revealed the presence of sequences complementary to this region. Transcriptional studies carried out in vitro indicated that this region is not transcribed under conditions that are optimal for utilization of the authentic rRNA promoter. Moreover, this region does not appear to form stable transcription complexes with RNA polymerase I transcription components. These data indicate that the mouse rDNA repeating unit differs from those of Xenopus spp. and Drosophila melanogaster in that reduplicated RNA polymerase I promoters are not found in the mouse rDNA spacer region.

Animals↗
PubMed1993

In situ blotting: a novel method for direct transfer of native proteins from sectioned tissue to blotting membrane: procedure and some applications.

We describe a novel technique for direct transfer of native proteins from unfixed frozen tissues sections to an immobilizing matrix, e.g., nitrocellulose, polyvinyliden difluoride, or positively charged nylon membranes. Proteins are directly blotted onto the membrane, providing optimal accessibility for molecular detection but retaining the anatomic localization at the cellular level. Within 10 min a maximum protein transfer is achieved independent of the protein molecular weight. The total protein bound was 80% of the maximal binding capacity of the blotting membrane and independent of the section thickness. These results indicate that the proteins that bind to the membrane originate from the cut cell monolayer that has direct contact with the blotting membrane. This in situ blotting method provides direct protein mapping from a single cell layer of a tissue section. The procedure includes cryosectioning at 20 microns and collecting sections on a dry blotting membrane at -20 degrees C. For protein transfer the blotted sections are thawed and incubated for 10 min with Tris buffer. After incubation the sections are removed from the membrane by high-pressure spray. The blotted membranes can be subjected to several detection assays. In the present study the presence of several proteins was demonstrated in brain and thymus by immunochemical and enzyme histochemical procedures.

Animals↗
PubMed1986

Cloning and characterization of a canine oral papillomavirus.

A papillomavirus, isolated from oral papillomas in young Beagles, was used to produce a live-virus vaccine. After the IM use of this vaccine, some dogs developed squamous cell carcinomas at the inoculation site. The virus was isolated from the original vaccine and was cloned into pBR322. A detailed restriction map of the viral genome was generated.

Animals↗
PubMed1995

Analysis of the neurofibromatosis 2 gene in human breast and hepatocellular carcinomas.

The neurofibromatosis 2 gene (NF2) mapped to chromosome 22q is a recently isolated tumor suppressor gene that participates in the tumorigenesis of cells of embryonic neural crest origin. The structural similarities between the NF2 product, merlin-schwannomin, and the band 4.1 family raise the possibility that merlin may participate in a wide range of cell activities, in which the cytoskeleton latticework has important roles to play. In order to examine the significance of NF2 in general carcinogenesis, comprehensive analyses of 68 cases of breast carcinoma, which shows frequent loss of heterozygosity at chromosome 22, and 48 hepatocellular carcinomas of different histological grade were performed. No mutation was detected by the polymerase chain reaction-single-strand conformation polymorphism method for six exons of NF2 in any of the cases examined, suggesting that NF2 may be less important in the tumorigenesis of breast and liver cancers than in that of cancers originating from the neural crest.

Base Sequence↗
PubMed2001

Genomic organization and chromosomal mapping of SPARC-like 1, a gene down regulated in cancers.

Human SPARC-like 1 (SPARCL1), also known as MAST9 or hevin, is a member of the SPARC protein family. Originally we identified SPARCL1 as one of the genes down regulated in human non-small cell lung cancer (NSCLC). Recent reports indicate that the down regulation of SPARCL1 also occurs in prostate and colon carcinomas, suggesting that SPARCL1 inactivation is a common event not only in NSCLCs but also in other tumors of epithelial origin. In the present work we report the cloning and mapping of the genomic locus of human SPARCL1. Using fluorescence in situ hybridization analysis, SPARCL1 was localized to chromosome 4q22-25, a region often deleted in human cancers. Furthermore, we show that the intron/exon organization of the human SPARCL1 gene is similar to its murine homologue SC1. SPARCL1 contains 11 exons and 10 introns which span approximately 47 kb of the genome. We also sequenced the 5'-flanking region of the human SPARCL1 gene containing 2.4 kb of the putative promoter region. The data presented herein are a prerequisite for deletion/mutation analysis of the SPARCL1 gene in tumors. In addition, knowledge of the SPARCL1 promoter sequence allows to investigate the regulation of SPARCL1 expression on the transcriptional level. Taken together our results will help to clarify the function of SPARCL1 in tumor formation.

Base Sequence↗
PubMed1999

Active role of a human genomic insert in replication of a yeast artificial chromosome.

Yeast artificial chromosomes (YACs) are a common tool for cloning eukaryotic DNA. The manner by which large pieces of foreign DNA are assimilated by yeast cells into a functional chromosome is poorly understood, as is the reason why some of them are stably maintained and some are not. We examined the replication of a stable YAC containing a 240-kb insert of DNA from the human T-cell receptor beta locus. The human insert contains multiple sites that serve as origins of replication. The activity of these origins appears to require the yeast ARS consensus sequence and, as with yeast origins, additional flanking sequences. In addition, the origins in the human insert exhibit a spacing, a range of activation efficiencies, and a variation in times of activation during S phase similar to those found for normal yeast chromosomes. We propose that an appropriate combination of replication origin density, activation times, and initiation efficiencies is necessary for the successful maintenance of YAC inserts.

Chromosomes, Artificial, Yeast↗
PubMed2002

Relationships of local inhibitory and excitatory circuits to orientation preference maps in ferret visual cortex.

The contribution and precise role of intracortical circuits in generating orientation tuned responses in visual cortical neurons is still controversial. To address this question, the relationship between excitatory and inhibitory synaptic connections and orientation maps in ferret striate cortex was investigated by combining in vivo optical imaging and in vitro scanning laser photostimulation. Excitatory and inhibitory inputs to pyramidal cells originated preferentially from regions with similar orientation preference. Prominent cross-orientation inhibition was not observed, arguing against cross-orientation models of orientation selectivity. The tuning of inhibitory inputs was significantly broader in both layer 2/3 and layer 5/6 pyramidal neurons compared to the tuning of excitatory inputs. Local excitatory inputs were more prominent in the 0-20 degrees tuning difference range between pre- and postsynaptic cells than inhibitory inputs, whereas inhibition dominated in the 20-40 degrees tuning difference range. These differences in tuning of excitatory and inhibitory inputs onto individual cells are consistent with the predictions of recurrent models of orientation selectivity.

Animals↗
PubMed1993

Deletion analysis of a replication origin of human cytomegalovirus by a novel assay system with a combination of microinjection and polymerase chain reaction.

We have developed a sensitive assay for DNA replication in mammalian cells that enables us to detect replicated DNA fragments of human cytomegalovirus. A 1/100 portion of DNA extracted from 1000 microinjected cells was subjected to polymerase chain reaction after digestion with appropriate restriction enzymes to differentiate replicated from nonreplicated plasmids. A portion of the amplified DNA was electrophoresed to detect replicated DNA. Subfragments of the HindIII fragment A (24 kb, map units 0.37-0.47) of strain Towne, which contains a previously identified origin of DNA replication (oriLyt), were analyzed by the assay system. A 4.3-kb subfragment (AatII-SacI) replicated as efficiently as the HindIII fragment A. Efficient replication ability was lost with a 1.3-kb deletion from the AatII end or a 0.9-kb deletion from the SacI end. These results suggest that the boundaries of oriLyt of Human cytomegalovirus strain Towne lie within the 1.3- and the 0.9-kb regions of the 4.3-kb fragment.

Base Sequence↗
PubMed1993

Branch duct origin of solid type pancreatic ductal carcinoma.

A case of branch duct origin of solid type pancreatic ductal carcinoma is presented. Stepwise tissue sections of an entire specimen, including the carcinoma area, and mapping, with special reference to the relationship between the main duct and the carcinoma area, were performed. Carcinoma cells of the branch duct showed intraductal spread with minimal stromal invasion. No carcinoma tissue was observed in the main duct. Therefore this case was considered to be of branch duct origin. The finding of histologically identified branch duct origin of solid type pancreatic ductal carcinoma indicates a distinct advantage to be gained by giving special attention to both the main and branch ducts when using image diagnosis for early detection of carcinomas, and may represent the first reported surgically resected case.

Humans↗
PubMed2002

CCR5 and HIV infection.

Chemokines and chemokine receptors play a crucial role in the trafficking of leukocyte populations across the body, and are involved in the development of a large variety of human diseases. CCR5 is the main coreceptor used by macrophage (M)-tropic strains of human immunodeficiency virus type 1 (HIV-1) and HIV-2, which are responsible for viral transmission. CCR5 therefore plays an essential role in HIV pathogenesis. A number of inflammatory CC-chemokines, including MIP-1 alpha, MIP-1 beta, RANTES, MCP-2, and HCC-1[9-74] act as CCR5 agonists, while MCP-3 is a natural antagonist of the receptor. CCR5 is mainly expressed in memory T-cells, macrophages, and immature dendritic cells, and is upregulated by proinflammatory cytokines. It is coupled to the Gi class of heterotrimeric G-proteins, and inhibits cAMP production, stimulates Ca2+ release, and activates PI3-kinase and MAP kinases, as well as other tyrosine kinase cascades. A mutant allele of CCR5, CCR5 delta 32 is frequent in populations of European origin, and encodes a nonfunctional truncated protein that is not transported to the cell surface. Homozygotes for the delta 32 allele exhibit a strong, although incomplete, resistance to HIV infection, whereas heterozygotes display delayed progression to acquired immunodeficiency syndrome (AIDS). Many other alleles, affecting the primary structure of CCR5 or its promoter have been described, some of which lead to nonfunctional receptors or otherwise influence AIDS progression. CCR5 is considered as a drug target in the field of HIV, but also in a growing number of inflammatory diseases. Modified chemokines, monoclonal antibodies and small chemical antagonists, as well as a number of gene therapy approaches have been developed in this frame.

Amino Acid Sequence↗
PubMed2002

Initiation sites are distributed at frequent intervals in the Chinese hamster dihydrofolate reductase origin of replication but are used with very different efficiencies.

Previous radiolabeling and two-dimensional (2-D) gel studies of the dihydrofolate reductase (DHFR) domain of Chinese hamster cells have suggested that replication can initiate at any one of a very large number of inefficient sites scattered throughout the 55-kb intergenic spacer region, with two broad subregions (ori-beta and ori-gamma) preferred. However, high-resolution analysis by a PCR-based nascent strand abundance assay of the 12-kb subregion encompassing ori-beta has suggested the presence of a relatively small number of fixed, highly efficient initiation sites distributed at infrequent intervals that correspond to genetic replicators. To attempt to reconcile these observations, two different approaches were taken in the present study. In the first, neutral-neutral 2-D gel analysis was used to examine replication intermediates in 31 adjacent and overlapping restriction fragments in the spacer, ranging in size from 1.0 to 18 kb. Thirty of 31 fragments displayed the complete bubble arcs characteristic of centered origins. Taking into account overlapping fragments, these data suggest a minimum of 14 individual start sites in the spacer. In the second approach, a quantitative early labeled fragment hybridization assay was performed in which radioactive origin-containing DNA 300 to 1,000 nucleotides in length was synthesized in the first few minutes of the S period and used to probe 15 clones distributed throughout the intergenic spacer but separated on average by more than 1,000 bp. This small nascent DNA fraction hybridized to 14 of the 15 clones, ranging from just above background to a maximum at the ori-beta locus. The only silent region detected was a small fragment lying just upstream from a centered matrix attachment region--the same region that was also negative for initiation by 2-D gel analysis. Results of both approaches suggest a minimum of approximately 20 initiation sites in the spacer (two of them being ori-beta and ori-gamma), with ori-beta accounting for a maximum of approximately 20% of initiations occurring in the spacer. We believe that the results of all experimental approaches applied to this locus so far can be fitted to a model in which the DHFR origin consists of a 55-kb intergenic zone of potential sites that are used with very different efficiencies and which are separated in many cases by a few kilobases or less.

Animals↗