Mapping of restriction sites in the transforming HpaI-E fragment of adenovirus type 5 DNA.

Adenovirus type 5 (Ad5) DNA was degraded with endo R . HpaI; the left-terminal fragment, HpaI-E has recently been shown to be the smallest segment of Ad5 DNA, that can transform non-permissive cells. This fragment was labelled at its termini by limited exonuclease III digestion followed by repair synthesis with DNA polymerase and alpha-32P-labelled deoxynucleoside triphosphates. It was then further digested with each of the restriction endonucleases HpaII, HaeIII, AluI, HinfI and TaqI; the cleavage products thus obtained were ordered into a physical map.

The nature of antibody heavy chain residue H6 strongly influences the stability of a VH domain lacking the disulfide bridge.

Monoclonal antibody mAb 03/01/01, directed against the musk odorant traseolide, carries a serine residue instead of the conserved Cys H92 in the heavy chain variable domain, and is thus lacking the highly conserved disulfide bridge. We investigated the energetic consequence of restoring the disulfide bond and the nature of residue H6 (Glu or Gln), which is poised to interact with Ser H92 in the recombinant scFv fragment obtained from this antibody. In the scFv fragment derived from this antibody, the stabilizing effect of Gln H6 over Glu was found to be as large as the effect of reintroducing the disulfide bond. We have analyzed the conformation and hydrogen bond pattern of Gln H6 and Glu H6 in antibodies carrying these residues and suggest mechanisms by which this residue could contribute to VH domain stability. We also show that the unpaired cysteine H22 is buried, and conforms to the expected VH structure. The antibody appears to have acquired two somatic mutations (Ser H52 and Arg H66), which had been previously characterized as having a positive effect on VH stability. The overall domain stability is the decisive factor for generating functional, disulfide-free antibody domains, and several key residues play dominant roles.

The nucleotide sequence of the transforming HindIII-G fragment of adenovirus type 5 DNA. The region between map positions 4.5 (HpaI site) and 8.0 (HindIII site).

The nucleotide sequence of the region between map positions 4.5 (HpaI-site) and 8.0 (HindIII-site) of adenovirus type 5 (Ad5) DNA has been determined. This stretch of DNA is part of the transforming HindIII-G fragment, which is 2809 nucleotides long. The sequenced segment was found to have a long open reading frame for protein biosynthesis, starting 23 nucleotides from the HpaI site and extending all the way to the HindIII-G site, which could code for a protein of at least 44 000 daltons. The possible correlation beteen the coding capacity of the HindIII-G fragment and the "transforming" proteins specified by it will be discussed in the light of the recent data on the splicing of early mRNAs.

Comparison of nucleotide sequences of the early E1a regions for subgroups A, B and C of human adenoviruses.

The early E1a regions (map position (0--4.5%) of highly oncogenic (subgroup A) adenovirus serotype 12 (Ad12), weakly oncogenic (subgroup B) adenovirus serotype 7 (Ad7) and non-oncogenic (subgroup C) adenovirus serotype 5 (Ad5) can convert cells in vitro into an immortal cell line. A comparison of the nucleotide sequences for the E1a regions of Ad12 (Sugisaki et al., 1980), Ad7 (Dijkema et al., 1980), and Ad5 (Van Ormondt et al., 1978) DNA is presented. The overall homology is about 50%, but certain segments of the E1a regions exhibit higher than average homology, for both coding and noncoding sequences. The latter may specify the initiation of DNA replication, DNA encapsidation and regulation of expression. The predicted polypeptides encoded by the E1a regions of the three serotypes exhibit a large degree of homology.

The nucleotide sequence of the transforming early region E1 of adenovirus type 5 DNA.

The sequence of the leftmost 11.3% of the non-oncogenic human adenovirus type 5 (Ad5) DNA has been determined. This segment contains the entire early region E1 of the Ad5 genome which has been shown to be involved in in vitro transformation of non-permissive rodent cells (Van der Eb et al., 1980). From the DNA sequence, and from the mRNA sequence data obtained by Perricaudet et al, (1979, 1980) for the E1 mRNAs from the closely related adenovirus type 2 (Ad2), it is possible to predict the primary structure of the polypeptides encoded by this region. The function of these proteins in cell transformation is discussed. From the positions of mapped restriction endonuclease sites and termini of RNA segments in the nucleotide sequence the length of the Ad5 DNA is estimated to be 36.6 kb.

The nucleotide sequence of adenovirus type 5 early region E1: the region between map positions 8.0 (HindIII site) and 11.8 (SmaI site).

The nucleotide sequence of the region between map positions 8.0 (HindIII site) and 11.8 (SmaI site) of adenovirus type 5 (Ad5) has been determined. Together with the sequences reported earlier (Van Ormondt et al., 1978; Maat and Van Ormondt, 1979) it encompasses the entire leftmost early region E1 of Ad5 DNA (4126 base pairs). The total sequence revealed a number of potential regulatory signals (promoter sites, ribosome binding sites, 3'-poly(A)-associated sequences), which confirm that region E1 is divided into subregions, E1a and E1b, and a region coding for semi-late viral protein IX. By taking into account the adenovirus 2 (Ad2) RNA-splicing data of Perricaudet et al. (1979; 1980) and the Ad2 RNA mapping data of Chow et al. (1979) we predict that E1a codes for polypeptides of 32, 26 and ca. 13 kd, and subregion E1b for polypeptides of 67 kd and 20 kd; the expected molecular weight of protein IX is 14.4 kd.

The nucleotide sequence of the transforming HpaI-E fragment of adenovirus type 5 DNA.

The primary structure of the HpaI-E fragment of adenovirus type 5 (Ad5) DNA has been determined, mainly by the method of Maxam and Gilbert (1977). This fragment comprises the leftmost 4.5% of the Ad5 genome, and has been shown to be the shortest DNA fragment capable of transforming cells. The identification of potential initiation and termination codons in the determined sequence indicates that two small polypeptides consisting of 186, and 81 amino acids, respectively, could be synthesized. Taking into account recent data on RNA splicing, a possibility is considered that this DNA may code also for larger polypeptides.

Gene organization of the transforming region of weakly oncogenic adenovirus type 7: the E1a region.

The structures of adenovirus type 7 (Ad7) cytoplasmic RNAs transcribed from the leftmost 4.5% of the viral genome during lytic infection of KB cells have been determined. The E1a region was found to specify three differently spliced mRNAs (I, II and III) which have common 5' and 3' termini. mRNAs I and II are transcribed between identical initiation and termination codons and code for polypeptides of 28 kd and 24 kd, whose only difference is an internal sequence of 32 amino acids present in the 28-kd protein. Translation of mRNA III initiates at the same AUG codon as in mRNA I and II, but uses a different reading frame beyond the splice point; consequently, it terminates at an earlier stop codon and yields a 6.3-kd polypeptide. Cytoplasmic E1a RNA was used as a template for in vitro protein synthesis in a cell-free system and found to encode polypeptides with apparent molecular weights of 42 kd, 40 kd, and 11 kd.

The nucleotide sequence of the gene for protein IVa2 and of the 5' leader segment of the major late mRNAs of adenovirus type 5.

We present here the primary structure of the region of human adenovirus 5 (Ad5) DNA from nucleotide 4001 through the HindIII site at nucleotide 6246 (map positions 0.11 to 0.17). The corresponding region in the closely related adenovirus type 2 (Ad2) encodes the spliced mRNA for viral protein IVa2 (Chow et al., 1979; Persson et al., 1979). Reverse transcription of the Ad5 pIVa2 mRNA localized the 5' terminus of the mRNA to approximately position 5840, and its splice coordinates to positions 5706 and 5427. From the data of Aleström et al. (1980) for Ad2, the 3' end of this mRNA was inferred to be specified by Ad5 nucleotide 4060. The nucleic acid data allow us to predict an Mr of 50873 for the IVa2 protein of Ad5, which is close to the experimentally determined value of 50000 (Persson et al., 1979). The DNA sequence described here also includes the information for the 5'-terminal leader segment of the major late mRNAs of Ad5.

The gene for polypeptide IX of human adenovirus type 7.

This paper describes the nucleotide sequence of subgroup B human adenovirus type 7 (Ad7) between positions 3351-4010, and of cloned cDNA derived from mRNAs encoded by this segment. One of these (mRNA VII) is shown to be unspliced, and has its 5'- and 3'-ends encoded by positions 3460 (determined by the nuclease S1 technique) and 3939, respectively. The mRNA sequence contains a single open reading frame for protein biosynthesis between the first available initiation triplet AUG at position 3481 to the stop codon UAA at position 3895. It can specify a polypeptide of 138 amino acids (14 098 daltons) which must be polypeptide IX of Ad7, as is evident from its mapping position, and from a comparison with the sequence of the protein IX gene of subgroup C adenovirus type 5.

Effect of a pmr 1 disruption and different signal sequences on the intracellular processing and secretion of Cyamopsis tetragonoloba alpha-galactosidase by Saccharomyces cerevisiae.

We fused the yeast-derived sequences encoding the invertase, acid phosphatase and alpha-factor pre- and prepro-signal peptides (SP) to the Cyamopsis tetragonoloba (guar plant) alpha-galactosidase(alpha Gal)-encoding gene and expressed these gene fusions in yeast. Whereas the amount of fusion protein produced by each of the constructs did not vary significantly, the secretion efficiency of the fusion protein that carried the SP of the prepro-alpha-factor (MF alpha 1) was consistently found to be about 10% higher than that of the other fusions (99% vs. 90%). Furthermore, when the secretion of alpha Gal was directed by the invertase (SUC2) SP, the intracellular enzyme localized to the endoplasmic reticulum (ER), whereas use of the MF alpha 1 SP caused the intracellular enzyme to be outer-chain-glycosylated and processed by the KEX2 endoproteinase, implying that it had passed the ER. These results suggest that the pro-peptide of MF alpha 1 stimulates the efflux of the heterologous protein from the ER. Null mutants of PMR1 (encoding a Ca(2+)-dependent ATPase) are known to give higher secretion efficiencies for a number of different heterologous proteins. Therefore, we also studied the secretion of alpha Gal in a pmr 1 disruption mutant. Structural analysis of the enzyme secreted by the mutant cells showed that it was completely processed by KEX2 and outer-chain-glycosylated, although the length of the outer-chain carbohydrate moiety was reduced when compared with the enzyme secreted by wild-type cells. These results contradict the hypothesis advanced by Rudolph et al. [Cell 58 (1989) 133-145] that disruption of PMR1 causes the secretory pathway to bypass the Golgi apparatus.

Cloning of cDNA encoding the sweet-tasting plant protein thaumatin and its expression in Escherichia coli.

The structural gene of the sweet-tasting plant protein (prepro)thaumatin was cloned and expressed in Escherichia coli. Expression was effected under control of lac and trp promoter/operator systems and through the use of bacterial ribosome-binding sites. The naturally occurring thaumatin II represents a processed form. The primary translation product, preprothaumatin, of the cloned mRNA-derived cDNA contains extensions at both the amino terminus and the carboxy terminus. The amino terminal extension of 22 amino acids is hydrophobic and very much resembles an excretion-related signal sequence. The six amino acids-long carboxy terminal extension is very acidic in character, in contrast to the overall highly basic thaumatin molecule. The possible role of such an acidic tail with respect to compartmentalization is discussed.

[A 61-year-old woman with an infected little finger]. / Een vrouw met een geïnfecteerde pink.

A 61-year-old woman was seen at the Emergency Department with a progressive infection of the little finger. A solid hemorrhagic bulla was seen with 2 central ulcers and the diagnosis 'ecthyma contagiosum' was made. This is a self-limiting infection caused by a parapoxvirus in sheep and can be transmitted to humans through direct contact with infected animals.

A method for sequencing restriction fragments with dideoxynucleoside triphosphates.

A rapid enzymatic approach is described for the sequence analysis of a 5' terminally labelled restriction fragment. It involves limited nicking of the strands of the molecule throughout the sequence by pancreatic DNAase I. The 3' hydroxyl groups exposed by each nick are then used to prime chain extension by DNA polymerase I in four separate reactions. Each reaction uses one of the four chain terminating dideoxynucleoside triphosphates (ddNT-PSs), together with the four deoxynucleoside triphosphates (dNTPs). In a single reaction all the 3' ends are terminated in positions of the same base, which is different for each of the four reactions. When the products of these reactions are resolved by gel electrophoresis according to size, a sequence can be deduced from the pattern of radioactive bands. Sequences can be determined onwards from 10-20 residues from the 5' labelled end. The length of sequence which can be determined is only limited by the resolution of the gel.

Evaluating the Cancer Information Service: a model for health communications. Part 1.

The Cancer Information Service (CIS) was established in 1975 by the National Cancer Institute (NCI) to meet the information needs of cancer patients, their families, health professionals, and the public. As the nation's foremost source for cancer information, the CIS applies a unique health communications model to bring the latest research findings on cancer prevention, detection, treatment, and supportive care to the nation. It does this through two main program components: a toll-free telephone service (1-800-4-CANCER) and an outreach program that focuses on providing technical assistance, specifically to partners reaching minority and underserved audiences. During its 22-year history, more than 7.5 million callers have reached the CIS telephone service. In addition, 100,000 requests are received each year from 4,500 organizations nationwide seeking cancer-related outreach expertise. This overview describes the CIS model for health communications, describes the program's impact in broad terms, and defines the critical role evaluation plays in each program component. The overview describes two customer satisfaction and impact surveys performed by an independent survey research firm in 1996 to evaluate the CIS model: (a) the telephone service user survey, a random sample of 2,489 persons representing major caller groups who were interviewed 3 to 6 weeks after their initial call to the CIS; and (b) the outreach partner survey, a random sample of 867 partner organizations, the majority of which reach minority and underserved audiences with information and programs, surveyed within a few months after a contact with the CIS outreach program. Impact data for both program areas were favorable: Approximately 8 out of 10 CIS callers reported that the information they received had a positive impact (either in eliciting a positive action [56%] or in reassurance of decisions made [22%]) and two-thirds of CIS partners said the CIS has an important impact on their programs.

Appendix to Part 1: Description of survey methods for the CIS telephone service user survey and CIS outreach partner survey.

Both the CIS telephone service user survey and CIS outreach partner survey were statistically based surveys conducted in accordance with standard research practices and techniques. Following are descriptions of the research designs for each survey, the sample selection techniques employed, and the response rate statistics.

Preparation and specificity of endonuclease IV induced by bacteriophage T4.

Bacteriophage-T4-induced endonuclease IV suitable for DNA sequence analysis has been prepared by a modified and easily reproducible method. The specificity of T4-induced endonuclease IV has been investigated in order to verify whether this enzyme exhibits a single nucleotide recognition or a short sequence recognition. The 5'-terminal dinucleotides and 3'-terminal nucleotides of oligonucleotides released by T4-induced endonuclease IV from three single-stranded DNAs (from bacteriophages phiX174, fd, M 13) have been analysed. In different DNAs, 74-82% of the 5'-terminal dinucleotides end in 5'-deoxycytidylic acid; small but significant levels of several dinucleotides ending in 5'-deoxyadenylic acid, 5'-thymidylic acid and 5'-deoxyguanylic acid are also found. As far as 3'-terminal nucleotides are concerned all nucleotides are present with a large predominance of thymidylic acid. It is concluded that T4-induced endonuclease IV recognizes short nucleotide sequences like all other DNases investigated so far. The spectrum of such sequences is, however, very narrow.

Primary care physicians' attitudes, knowledge, and practices related to cancer clinical trials.

BACKGROUND: Participation of patients in cancer clinical trials is disappointingly low and several physician-based factors are thought to be responsible. METHODS: In 1998-1999, the National Cancer Institute (NCI) conducted a probability survey of three primary care physician groups to gain a better understanding of the barriers to clinical-trial patient accrual from their perspective. RESULTS: Findings from this survey of 706 primary care physicians indicate that the vast majority (98%) refer their patients with cancer to a specialist for cancer treatment and rarely bring up the topic of cancer clinical trials. Frequently cited reasons for not mentioning clinical trials are preferring to leave that discussion to the oncologist (41%) and being unaware of any clinical trials that may be available for the patient (37%). CONCLUSION: Primary care physicians may represent an important untapped resource for introducing the concept of clinical trials as an option to newly diagnosed cancer patients.

Monitoring water content in deforming intervertebral disc tissue by finite element analysis of MRI data.

Mechanical loading, occurring during normal daily life, causes fluid to be expelled from intervertebral discs. Excessive fluid loss during heavy loading might make the disc more vulnerable to damage. In this study, fluid loss was investigated in vitro through monitoring the loss of MRI signal intensity in four bovine coccygeal intervertebral discs, compressed at 2000 N during 1.5 hr. The MRI signals were analyzed with the aid of finite element models to account for the deformation of the tissue. A gradual signal loss over time was found during loading, the most pronounced loss occurring in the central disc region. Initial patterns of signal distribution were quite variable between specimens but repeatable within specimens.