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Simultaneous manipulation of propositional and analog codes in picture memory.

Revealing a picture as a whole or as momentarily displayed strips presumably manipulated the propositional coding of the picture. Analog manipulation employed superimposition of a grid pattern. Effectiveness of simultaneous manipulations was tested via recognition with three foil categories, each of which presumably tapped the analog or propositional codes mainly, or both of them in detection. While figural units' perspective reversals should presumably be detected via the analog code, their locational displacements should be detected via the propositional code. Foils with novel item substitution should be detected by both codes. On the basis of such presumptions about encoding and recognition manipulations, certain predictions were made for scan and grid effects in "reversal," "displacement," and "novelty" foil categories. Full confirmation of these predictions was then taken as evidence that both analog and propositional codes do exist. Once such evidence was established, other issues were tackled, showing that target detection involves the propositional code only, while foil detection involves both codes. Further, recognition hypermnesia, where performance improves with repeated testing, was obtained only when both codes were attenuated. Data suggested that subjects might be switching from analog to propositional codes when both codes were strong, thereby preventing hypermnesia.

Adult

Molecular cloning and sequencing of a cDNA encoding the acyl carrier protein and its flanking domains in the mammalian fatty acid synthetase.

Cloned cDNAs containing coding sequences for domains proximal to the carboxy terminus of the rat fatty acid synthetase have been isolated using an expression vector and domain-specific antibodies. The coding regions were assigned to specific domains of the multifunctional complex by identification of sequences coding for characterized peptide fragments and by recognition of sequences homologous to other monofunctional enzymes. Two clones contain the entire coding region for the acyl carrier protein domain. The sequence is flanked at the 3'-end by a region coding for the thioesterase domain and at the 5'-end by a sequence coding for a reductase, most likely the ketoreductase domain. Thus the ordering of these domain-coding regions in the fatty acid synthetase mRNA is established. The acyl carrier protein domain exhibits about 25% homology with that of the discrete monofunctional acyl carrier proteins of Escherichia coli, spinach and barley, the ketoreductase domain exhibits about 25% homology with bacterial dihydrofolate reductases and the active site of the thioesterase domain exhibits both primary and secondary structural features common to the serine proteases. These findings lend support to the hypothesis that the polyfunctional fatty acid synthetase probably arose by a complex evolutionary process involving fusion of genes coding for seven individual enzymes.

Acyl Carrier Protein

A possible model for cell-cell recognition via surface macromolecules.

Alternative possibilities for the establishment of the proper cell distribution during embryogenesis are summarized at the beginning, followed by an assessment of the examples known so far where cell-cell recognition is known to be mediated via cell surface components. In the second part the species-specific recognition process which occurs during the sorting-out of dissociated sponge cells is analysed since it may serve as a possible model for cell-cell recognition in higher animals. Three possible mechanisms for the establishment of proper cell distribution are considered. These include, first, chemotaxis: secondly, guidance of cell or cell sheet movement by extracellular matrix or by surrounding cells and thirdly, random movement followed by recognition at the final point of destination. Recognition is necessary for both of the two latter processes, i.e. for cell guidance as well as for locking the cells into their final position after random movement. Two basically different recognition mechanisms should be distinguished from each other. On the one hand cells may recognize each other with the help of macromolecules situated in or just outside of the plasmamembrane which fit to each other like enzymes and substrates or antibodies and antigens. On the other hand, cells may exchange information by exchanging cytoplasmatic components via vesicles or gap junctions. The species-specific aggregation of dissociated sponge cells is considered to be a possible model for cell-cell recognition in higher animals. A proteoglycan-like intercellular macromolecule called aggregation factor seems to mediate recognition of a given species of cells in the reaggregation process of dissociated cells. The data available at the present time suggest that a monovalent surface macromolecule (baseplate) may mediate the recognition process probably by recognizing the carbohydrate side chains of the multivalent proteoglycan aggregation factor. A cell-free system was devised to mimic this aggregation process. Addition of aggregation factor to baseplate-coated sepharose beads of approximately the size of the original sponge cells has essentially the same characteristics as the cellular system. Macromolecule-coded surface information for the recognition between cells has not been established during the embryogenesis of higher animals and remains an interesting challenge.

Animals

Coding coenzyme handles: a hypothesis for the origin of the genetic code.

The coding coenzyme handle hypothesis suggests that useful coding preceded translation. Early adapters, the ancestors of present-day anticodons, were charged with amino acids acting as coenzymes of ribozymes in a metabolically complex RNA world. The ancestral aminoacyl-adapter synthetases could have been similar to present-day self-splicing tRNA introns. A codon-anticodon-discriminator base complex embedded in these synthetases could have played an important role in amino acid recognition. Extension of the genetic code proceeded through the take-over of nonsense codons by novel amino acids, related to already coded ones either through precursor-product relationship or physicochemical similarity. The hypothesis is open for experimental tests.

Amino Acyl-tRNA Synthetases

Use of document image processing in cancer registration: how and why?

The aims of the study are to test and evaluate a system for document image processing (DIP) in cancer registration and coding. The results from use of optical character recognition (OCR) and image character recognition (ICR) in the data entry process are of particular interest. Cases of cancer must be reported both by clinicians and pathologists. Annually the Registry receives 80,000 reports concerning new cases and supplementary information on patients who are registered earlier [1]. Clinicians report new cancer cases to the Cancer Registry on a special designed form. Optionally, they can use a software-based application delivered from the Registry free of charge. As a part of the DIP-system, an optical digital image processing text (ODIT) system is used in recognition of machine- and hand-printed characters. The traditional registration and coding system is run parallel to the new system. After a period of testing the DIP-system nationwide, the new system will be evaluated and compared to the traditional system. The first part of the study compared the results from three character readers (CGK, XDR, and Nestor), which have been tested on our own application-specific data in a realistic setting [2]. The test shows that over 90% of the digits and about 70% of the letters can be correctly recognized by the system [3]. Feedback is communicated to clinicians to improve the quality of hand-print in the forms. It is to be hoped that this action among others will give a better recognition result for the next part of the study. The XDR Network Reader will serve as a reader in the ODIT-system chosen for the rest of the study. The software system Open Image Link shall serve as the image manager. De facto standard software like MS-Access is used as a part of the DIP-system. The next part of the study is the main project and started at the end of June 1994. Results from the project will be presented. Does the new system produce the expected high percentage of the clinicians will optionally use the software application instead of of paper forms in reporting cancer? And at what quality of data? Will the new DIP-system be cost-effective? The results from the study and the reasons for running such a project will be discussed.

Medical Oncology

How Cro and lambda-repressor distinguish between operators: the structural basis underlying a genetic switch.

Knowledge of the three-dimensional structures of the lambda-Cro and lambda-repressor proteins in complex with DNA has made it possible to evaluate how these proteins discriminate between different operators in phage lambda. As anticipated in previous studies, the helix-turn-helix units of the respective proteins bind in very different alignments. In Cro the recognition helices are 29 A apart and are tilted by 55 degrees with respect to each other, but bind parallel to the major groove of the DNA. In lambda-repressor [Beamer, L. J. & Pabo, C. O. (1992) J. Mol. Biol. 227, 177-196] the helices are 34 A apart and are essentially parallel to each other, but are inclined to the major grooves. The DNA is much more bent when bound by Cro than in the case with lambda-repressor. The first two amino acids of the recognition helices of the two proteins, Gln-27 and Ser-28 in Cro, and Gln-44 and Ser-45 in lambda-repressor, make very similar interactions with the invariant bps 2 and 4. There are also analogous contacts between the thymine of bp 5 and, respectively, the backbone of Ala-29 of Cro and the backbone of Gly-46 of lambda-repressor. Otherwise, however, unrelated parts of the two proteins are used in sequence-specific recognition. It appears that similar contacts to the invariant or almost invariant bps (especially 2 and 4) are used by both Cro and lambda-repressor to differentiate the operator sites as a group from other sites on the DNA. The discrimination of Cro and lambda-repressor between their different operators is more subtle and seems to be achieved primarily through differences in van der Waals contacts at bp 3', together with weaker, less direct effects at bps 5' and 8', all in the nonconsensus half of the operators. The results provide further support for the idea that there is no simple code for DNA-protein recognition.

Bacteriophage lambda

T cells and human autoimmune thyroid disease: emerging data show lack of need to invoke suppressor T cell problems.

Human T cells recognize self and foreign antigens when such antigens are processed into small peptides and bound to molecules coded for by genes of the HLA region on chromosome 6. The part of the T-cell surface which is responsible for such recognition is a set of molecules coded for by a variety of genes and known as the T-cell-receptor complex. In animal models, T cells are able to transfer autoimmune thyroiditis and T cells have, therefore, long been implicated in the etiology of human autoimmune thyroid disease (AITD). Information gained from the study of intrathyroidal T cells and thyroid antigen-specific T-cell clones has shown that in patients with Graves' disease, mainly helper T-cell clones have been obtained, whereas in autoimmune (Hashimoto's) thyroiditis cytolytic T-cell clones may be predominant. Such thyroid antigen-specific T cells have now been shown to recognize one or other of the three major thyroid-specific antigens; thyroglobulin, thyroid peroxidase, or the TSH receptor and efforts are currently in progress to characterize the T-cell epitopes of these major thyroid autoantigens. Recent findings of restricted T-cell receptor V gene use amongst intrathyroidal T cells confirm the primary role of T cells in human thyroid autoimmune processes leading to AITD. However, the mechanisms whereby such autoreactive T cells escape deletion and anergy, and how they become activated, remain uncertain. There is compelling evidence that the thyroid cell itself, by expressing HLA molecules, and presenting antigen directly to the T cells, may initiate disease, perhaps after an external insult.

Amino Acid Sequence

The Gene-Finder computer tools for analysis of human and model organisms genome sequences.

We present a complex of new programs for promoter, 3'-processing, splice sites, coding exons and gene structure identification in genomic DNA of several model species. The human gene structure prediction program FGENEH, exon prediction-FEXH and splice site prediction-HSPL have been modified for sequence analysis of Drosophila (FGENED, FEXD and DSPL), C.elegance (FGENEN, FEXN and NSPL), Yeast (FEXY and YSPL) and Plant (FGENEA, FEXA and ASPL) genomic sequences. We recomputed all frequency and discriminant function parameters for these organisms and adjusted organism specific minimal intron lengths. An accuracy of coding region prediction for these programs is similar with the observed accuracy of FEXH and FGENEH. We have developed FEXHB and FGENEHB programs combining pattern recognition features and information about similarity of predicted exons with known sequences in protein databases. These programs have approximately 10% higher average accuracy of coding region recognition. Two new programs for human promoter site prediction (TSSG and TSSW) have been developed which use Gosh (1993) and Wingender (1994) data bases of functional motifs, respectively. POLYAH program was designed for prediction of 3'-processing regions in human genes and CDSB program was developed for bacterial gene prediction. We have developed a new approach to predict multiple genes based on double dynamic programming, that is very important for analysis of long genomic DNA fragments generated by genome sequencing projects. Analysis of uncharacterized sequences based on our methods is available through the University of Houston, Weizmann Institute of Science email servers and several Web pages at Baylor College of Medicine.

Animals

Immunoglobulin variable region heptamer-nonamer recognition sequence joined to rearranged D-J segment: implications for the immunoglobulin recombinase mechanism.

We have found a novel immunoglobulin gene rearrangement in a murine hybridoma in which a heavy chain variable region (VH) heptamer-nonamer recognition sequence is joined to the diversity segment (D) through head-to-head fusion. The heptamer-nonamer recognition sequence and its adjacent 5' DNA are derived from the downstream flanking region of a germline VH gene. Sequence analysis indicates that this adjacent DNA is homologous to the downstream flank of VH108B, and it has characteristics of RNA processing that may suggest it was derived from an mRNA intermediate; these unusual features indicate that the segment is a processed gene. Because of head-to-head fusion, the recognition sequence and the flanking sequence are in opposite transcriptional polarity to D. The latter is joined correctly at its 3' border to a joining (J) gene segment. A gamma 1 constant region (but not mu) is located further downstream. Thus this fragment has several features common to normal immunoglobulin heavy chain gene rearrangement despite the unusual joining event involving V-D. Linkage of the VH heptamer-nonamer recognition sequence to D has not been observed previously. Although the recognition sequence described is inverted with respect to D and J, the endonucleolytic process that cleaved the recognition sequence at the 5' border of the heptamer before rearranging it to D was accurate. We suggest that of the three functions associated with the recombinase reaction; recognition, cutting, and ligation, only recognition and cutting may be limited to specific structures, and the ligation step may be less restricted because it is not confined to forming coding-to-coding or flank-to-flank joints. This aberrant ligation product suggests that the information leading to normal rearrangements may be found in structures that include more than the recognition sequences or coding regions alone, because the joining described here has spliced the incorrect end of a recognition sequence to a coding region to yield a nonproductive recombination.

Animals

The sequence of a 27 kb segment on the right arm of chromosome VII from Saccharomyces cerevisiae reveals MOL1, NAT2, RPL30B, RSR1, CYS4, PEM1/CHO2, NSR1 genes and ten new open reading frames.

The DNA sequence of a 26,677 bp fragment from the right arm of chromosome VII from Saccharomyces cerevisiae reveals 18 open reading frames (ORFs) longer than 300 bp. Eight ORFs correspond to previously characterized genes. G6620 is the 3' end of the MOL1 gene coding for a polypeptide similar to stress-inducible proteins from Fusarium; G6630 is the NAT2 gene which encodes a methionine N-acetyltransferase; G6635 is the RPL30B gene coding for the ribosomal protein L30; G6658 is RSR1 encoding a ras-related protein; G6667 is CYS4, the gene for cystathionine beta-synthase; G6670 is identical to ORF2 located close to CYS4; G6673 is PEM1/CHO2 encoding a phosphatidylethanolamine methyltransferase; G7001 is the NSR1 gene coding for a nuclear signal recognition protein. G6664 shares significant homology with the ORF YKR076w from chromosome XI. The other nine ORFs show no significant homology to any protein sequence presently available in the public data bases.

Amino Acid Sequence

DNA synthesis in CD4- and CD8-positive cells in synovial fluid of patients with reactive and rheumatoid arthritis.

The occurrence of MHC class I antigens and microbial antigens derived from the triggering infection of the diseased joints in reactive arthritis (ReA) seems to set the stage for local immune activation. In this report activated lymphocytes are demonstrated by using an avidin-biotin-peroxidase complex (ABC) method combined with autoradiography that identifies DNA synthesis and, thus, activation. Most of the activated T lymphocytes in reactive arthritis were found to belong to the CD8 suppressor/cytotoxic T-lymphocyte subset. In striking contrast, the majority of the activated T lymphocytes detected in rheumatoid arthritis (RA) synovial fluid belonged to the CD4 helper/inducer subset. These findings agree well with the assumption that CD8-positive cells identify the foreign antigen in the context of class I antigens, whereas CD4-positive cells are found to be associated with the recognition of MHC locus II coded HLA antigens.

Adult

Expression, purification, and use as an antigen of recombinant sugarcane mosaic virus coat protein.

A high titre (1:10,000) antiserum was raised in a rabbit against the coat protein of sugarcane mosaic potyvirus (SCMV), by injecting a preparation of recombinant coat protein purified from a fusion protein expressed in E. coli. The fusion protein consisted of the MalE maltose binding protein (MBP) and the viral coat protein separated by the protease factor Xa cleavage site. The fusion protein was encoded by the plasmid pMAL-cCPM, which was constructed by cloning a modified coat protein gene to the 3' end of the MBP/factor Xa coding region. The coat protein gene was modified by site-directed mutagenesis so that the ATG start codon in the original construct was replaced by the codon AGC, deleting the NcoI restriction site (C/CATGG) and creating a unique Eco47III site (AGC/GCT). Endonuclease restriction with Eco47III resulted in a DNA fragment with GCT as the first three nucleotides. This triplet encodes alanine, which is the proposed N-terminal amino acid residue of the mature native coat protein. This modified coat protein coding region was ligated directly behind the nucleotide code for the amino acid recognition sequence for factor Xa. Expression was induced with IPTG and the recombinant fusion protein was extracted from the bacterial lysate by amylose resin column affinity chromatography and the two domains separated by factor Xa proteolysis. The coat protein was then purified from the maltose binding protein by ion exchange chromatography in buffer containing 6 M urea. A highly purified sample which contained 150 micrograms of both full-length and truncated coat proteins, was recovered from a litre of bacterial broth. The antiserum reacted with native coat protein in SCMV-infected sugarcane, and with recombinant coat proteins expressed in E. coli and sugarcane protoplasts with little or no cross-reaction with sugarcane proteins.

Amino Acid Sequence

Mutagenic effects of restriction enzymes in Chinese hamster cells: evidence for high mutagenicity of Sau3AI at the hprt locus.

CHO cells were exposed to seven different restriction endonucleases by electroporation and their cytotoxicity and mutagenicity measured. Cell killing as determined by a colony formation assay occurred in a concentration-dependent manner for each enzyme. The D0 of the survival curves were: MspI = 24U; AluI = 31U; Sau3AI = 106U; HaeIII = 46U; HinfI = 30U; PvuII = 35U; BamHI = 163U. BamHI and Sau3AI were particularly ineffective in cell killing. For the 6-base recognition sequence enzymes, PvuII (a blunt-ended cutter) was much more cytotoxic per unit electroporated than BamHI (a sticky-ended cutter). Among the 4-base cutters, Sau3AI and HaeIII were generally less cytotoxic than HinfI or PvuII. Cell killing appeared to depend on the nature of the recognition sequence and cutting sites rather than on the cutting frequency. The mutagenic effects of these restriction endonucleases were investigated by measuring the induced frequencies of hprt gene mutations. The mutagenicity of Sau3AI was dramatically higher than the other enzymes, increasing linearly with dose up to 35U. When normalized for survival, the mutagenicity of Sau3AI relative to the other enzymes was even greater. The mutagenic effect of BamHI, which has the same 5' protruding site as Sau3AI, was much lower at similar dose and survival levels. MspI, BamHI, and PvuII which have no recognition sites within the hprt coding sequence were marginally- or non-mutagenic. Based on these results and the distribution of cutting sites within the hprt cDNA for the enzymes studied, the hypothesis is discussed that a region in exon 4 is highly sensitive to the induction of mutants by DNA double-strand breaks.

Animals

Interferon effect on ribosomal ribonucleic acid related to chromosome 21 ploidy.

Antiviral and cell-growth-inhibitory activities of human interferon were shown to be related to the activity of a gene or genes present on chromosome 21. The 18s rRNA is vital to cell growth; it is capable of a viral-mRNA-recognition function and it is coded for by genes a portion of which are present on chromosome-21. A previously reported ability of human interferon to affect rRNA metabolism is characterized by a decrease in the sucrose-gradient-peak ratio of radiolabelled 28S to 18S rRNA in extracts from the cytoplasm of interferon-treated human fibroblasts. In the present report, interferon dose-response curves are presented demonstrating a direct relationship between a decrease in this ratio and interferon concentrations in the media. By using this virus-independent cytoplasmic rRNA assay, eight human fibroblast lines, differing in chromosome 21 ploidy, were tested for sensitivity to human interferon. Two monosomy-21, two euploid-21 and four trisomy-21 cell lines were tested. The monosomy-21 cell populations were significantly less sensitive to interferon than the other six cell types tested. Of the cell lines tested, the most sensitive, by a wide margin, was a trisomy-21 line. Trisomy-21 cell monolayer sensitivity, however, varied widely within the range from normal to supersensitive. These observations suggest that interferon's ability to affect rRNA metabolism is related to the activity of a gene or genes present on chromosome 21.

Cell Line

[Transcranial color-coded duplex ultrasound in interventional therapy of cerebral aneurysms. A pilot study].

PURPOSE: To assess the diagnostic potential of transcranial colour-coded duplex sonography in the recognition of residual perfusion of coiled cerebral aneurysms. METHOD: 10 patients (7 female, 3 male) with angiographically verified cerebral aneurysms (basilar artery n = 2, mean size 8.5 mm; supraclinoid internal cerebral artery n = 2, mean size 15.5 mm; posterior communicating artery n = 2, mean size 7 mm; anterior communicating artery n = 2, mean size 4.5 mm) were examined by TCCD using a 2.25 MHz probe immediately following embolisation and 12-24 hours prior to angiographic evaluation. RESULTS: 9 patients had a sufficient temporal bone window. Transtemporal localization of the embolised aneurysms was feasible in 7 of the 9 cases. One partially thrombosed aneurysm of the basilar artery (1 mm) and one completely thrombosed aneurysm of the anterior communicating artery could not be visualised. In 7 cases no discrepancies between TCCD and angiography were evident, since rest, perfusion (n = 6) and complete thrombosis were unanimously diagnosed as such. Mean size of the rest perfused lumina was 4.4 mm (minimum 1.5 mm; maximum 8 mm). Platinum coils appeared as hyperechogenic lesions with an echogenicity comparable to the skull. CONCLUSION: With TCCD residual perfusion of coiled aneurysms was feasible in most cases. The method seems to be specific but not sensitive in the detection of incompletely coiled aneurysms. Limitations are insufficient transtemporal bone windows and inaccessible aneurysm location, especially the anterior communicating artery.

Adult

Expression of the sis gene by endothelial cells in culture and in vivo.

Recognition that the sis gene codes for a protein homologous with at least one of the two chains of platelet-derived growth factor has made it possible to directly assess transcriptional expression of platelet-derived growth factor both in cultured cells and in tissue obtained in vivo. We have found that a 3.7-kilobase RNA homologous to the sis gene is expressed at moderate levels in cultured human and bovine endothelial cells, at low levels in in vivo endothelium from human umbilical vein, and at very low levels in bovine aortic endothelium in vivo. This RNA migrates at the same rate as the previously reported sis band in the HUT 102 human T-cell lymphoma line. This band is not found in RNA extracted from freshly obtained bovine aortic media or from human foreskin fibroblasts or cultured fetal human aortic smooth muscle cells. Our in vitro results suggest that the sis gene is responsible for at least part of the platelet-derived growth factor-like mitogenic activity secreted by cultured endothelial cells and indicate that the sis gene is readily activated in endothelial cells during the transition from in vivo conditions to in vitro growth as a monolayer on plastic. Expression of the sis gene by endothelium in vivo raises the possibility that platelet-derived growth factor has a role in the development of the vascular system in the young animal and in the maintenance of the normal vascular system in the adult.

Animals

A peptide to DNA conversion program.

A modification and extension of the computer program REVCUT (Blumenthal et al, Nucl. Acids Res. 10, 91-101 (1982) is described. The new program searches for restriction endonuclease recognition sites that are not coding DNA sequences of a protein of known aminoacid sequence using bit patterns. The modifications make the program more accurate and extend the range of the restriction endonucleases.

Amino Acid Sequence

Characterization of a binding site for the herpes simplex virus type 1 UL9 origin-binding protein within the UL9 gene.

Gene UL9 of herpes simplex virus type 1 (HSV-1) encodes a sequence-specific origin-binding protein (OBP) that plays a direct and essential role in viral DNA synthesis. A search of the complete HSV-1 genomic sequence for possible OBP binding sites lying outside the known origins of replication revealed the presence of a very close match to the OBP recognition sequence within the UL9 coding region. The ability of OBP to bind to this site (referred to as the 'UL9 box') was confirmed by DNase I footprinting and gel retardation assays, and filter binding experiments demonstrated that the affinity of OBP for the UL9 box was of the same order as for its high affinity sites within the three replication origins. To investigate whether binding of OBP to the UL9 box played a role during viral replication we constructed a mutant virus in which the sequence was altered in such a way as to preserve the encoded amino acid sequence whilst abolishing the ability of OBP to bind. Growth of the virus was indistinguishable from wild-type and no alterations were observed in the accumulation of transcripts from the UL9 region of the genome. In addition, a DNA fragment containing the UL9 box sequence did not exhibit origin activity in a transient assay for viral DNA synthesis. We therefore conclude that binding of OBP to the UL9 box is not essential for virus growth and that expression of the UL9 gene is unlikely to be autoregulated through this site.

Base Sequence