Biomedical subjects
M J Braun
Publications and source records attributed to M J Braun.
Clinical evaluation of a high-resolution new peripheral quantitative computerized tomography (pQCT) scanner for the bone densitometry at the lower limbs.
Precision, long-term stability, linearity and accuracy of the x-ray peripheral quantitative computerized tomographic (pQCT) bone scanner XCT 3000 (Norland-Stratec Medical Sys.) were evaluated using the European Forearm Phantom (EFP). In vivo measurements were assessed using a standardized procedure at the distal femur and the distal tibia. In the patient-scan mode, the spatial resolution of the system was 1.04 +/- 0.05 lp/mm as measured at the 10% level of the modulation transfer function (MTF). The contrast-detail diagram (CDD) yielded a minimal difference in attenuation coefficient (AC) of 0.07 cm(-1) at an object size of 0.5 mm. The effective dose for humans was calculated to be less than 1.5 microSv per scan. Short-term precision in vivo was expressed as root mean square standard deviation of paired measurements of 20 healthy volunteers (RMSSD = 0.5%). At the distal femur total volumetric density (ToD) and total cross-sectional area (ToA) were found to be less sensitive to positioning errors than at the distal tibia. Structural parameters like the polar cross-sectional moment of inertia (CSMIp) or the polar cross-sectional moment of resistance (CSMRp) showed a good short-term precision at the distal femur (RMSSD = 1.2 and 1.4%). The relation between the two skeletal sites with respect to CSMIp or CSMRp showed a high coefficient of determination (r2 = 0.77 and 0.74).
A molecular phylogenetic survey of the nightjars and allies (Caprimulgiformes) with special emphasis on the potoos (Nyctibiidae).
A 656-pb fragment of the mitochondrial cytochrome b gene was sequenced for six species of Central and South American potoos (genus Nyctibius, Nyctibiidae) as well as for selected representatives of all other caprimulgiform families. Sequence divergence among potoos was much higher (11.1-16.2%) than has typically been observed among congeneric species of birds, suggesting that the species of Nyctibius are quite old. Divergence among families was also quite high (13.7-21.8%), confirming recent DNA-DNA hybridization studies. Such high genetic divergences in a functionally constrained gene like cyt b indicate that many of those sites which are free to vary will have experienced multiple substitutions. We therefore performed phylogenetic analysis using parsimony under a variety of weighting schemes intended to reduce the effect of multiple substitutions. The monophyly of all the traditional caprimulgiform families was confirmed and a number of new hypotheses of relationship emerged. From our analysis, it appears that the oilbird (Steatornis) is an ancient member of the order, and it is not closely related to the potoos. We also note the close link between Aegothelidae and Caprimulgidae, and the basal position of the Podargidae/Batrachostomidae clade in the phylogeny. These results are in accordance with several classical works of the first half of the century. The relationships of the various Nyctibius species to each other have not been fully resolved; however, a close link between N. leucopterus and N. maculosus appears to be highly probable from our data.
Allelic diversity in alligator microsatellite loci is negatively correlated with GC content of flanking sequences and evolutionary conservation of PCR amplifiability.
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Increasing proportions of uracil in DNA substrates increases inhibition of restriction enzyme digests.
Techniques that rely upon the incorporation of uracil into DNA are being published with increasing frequency, especially in PCR protocols. We report here the efficiency of 18 type II restriction enzymes to digest PCR amplicons synthesized with varying proportions of TTP to dUTP in the PCR mixture. We find that most enzymes with A:T/U bp in their recognition site digest the amplicons less efficiently as the percentage of dUTP in the reaction mixture is increased. This effect is most dramatic when the proportion of dUTP in the nucleotide mixture exceeds 50%. All but one of the enzymes which fail to digest amplicons that are synthesized with 100% dUTP digest some amplicons which are synthesized with 90% dUTP.
Equipping and organizing comparative molecular genetics laboratories.
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Use of randomly amplified polymorphic DNA markers in comparative genome studies.
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Random amplified polymorphic DNA (RAPD) markers readily distinguish cryptic mosquito species (Diptera: Culicidae: Anopheles).
The usefulness of random amplified polymorphic DNA (RAPD) was examined as a potential tool to differentiate cryptic mosquito species. It proved to be a quick, effective means of finding genetic markers to separate two laboratory populations of morphologically indistinguishable African malaria vectors, Anopheles gambiae and An. arabiensis. In an initial screening of fifty-seven RAPD primers, 377 bands were produced, 295 of which differed between the two species. Based on criteria of interpretability, simplicity and reproducibility, thirteen primers were chosen for further screening using DNA from thirty individuals of each species. Seven primers produced diagnostic bands, five of which are described here. Some problematic characteristics of RAPD banding patterns are discussed and approaches to overcome these are suggested.
Phylogenetic relationships of the thylacine (Mammalia: Thylacinidae) among dasyuroid marsupials: evidence from cytochrome b DNA sequences.
DNA sequences from the mitochondrial cytochrome b gene were obtained from a museum specimen of the presumed extinct thylacine (Thylacinus cynocephalus) and were compared with homologous sequences from 13 representatives of the Australian marsupial family Dasyuridae. The relationship of the thylacine to dasyurids has been suggested by previous anatomical and molecular studies, but its position within the dasyuroid radiation has not been addressed with genetic data. Phylogenetic analysis of the sequences reported here suggests that the thylacine is a sister group to Dasyuridae and lends support to the hypothesis that Thylacinus represents an ancient Australian marsupial lineage. Relationships with Dasyuridae support the results of other recent molecular studies, particularly in showing the affinities of endemic New Guinean subfamilies with larger Australian clades.
Nucleotide sequence and genome organization of biologically active proviruses of the bovine immunodeficiency-like virus.
The complete nucleotide sequences and translations of major open reading frames (ORF) of two distinct, infectious, proviral molecular clones (106 and 127) of the bovine immunodeficiency-like virus (BIV), obtained from a single virus isolation, were determined and compared. The genomes of BIV 127 and 106 are 8482 and 8391 nucleotides (nt), respectively, in the form predicted for the viral RNA. The structural organization of the genomes of BIV 127 and 106 are identical to one another and most similar to that of the lentivirus subfamily of retroviruses. In addition to gag, pol, and env genes, the BIV genome contains five short ORFs between and overlapping pol and env in the "central region," a hallmark of the lentiviruses which is believed to play an important role in their pathogenesis. Three of the short ORFs in the central region of BIV have been identified by location and structural similarity to the nonstructural/regulatory genes (vif, tat, and rev) of other lentiviruses; we also discovered two unique ORFs, termed W and Y, which may serve as exons for novel genes. BIV does not have the nef gene found in primate lentivirus genomes. The proviral LTR of BIV 127 is 589 nt, contains regulatory signals for initiation, enhancement, and termination of viral transcription, and has sequences related to the Sp1 and NF-kappa B binding sites. A major deletion (87 nt) in the env gene and 2 minor deletions (2 nt each) in the R regions of the LTRs account for the smaller size of clone 106. Numerous point mutations were also present; some caused coding substitutions that were most prevalent in the env encoding ORF. These data suggest that, within a single virus isolate, BIV displays extensive genomic variation. These infectious clones of BIV represent well-defined tools with which to analyze the function of the various ORFs and to dissect the molecular mechanisms of replication and pathogenesis.
Stability of RNA stem-loop structure and distribution of non-random structure in the human immunodeficiency virus (HIV-I).
The stability of potential RNA stem-loop structures in human immunodeficiency virus isolates, HTLV-III and ARV, has been calculated, and the relevance to the local significant secondary structures in the sequence has been tested statistically using a Monte Carlo simulation method. Potentially significant structures exist in the 5'non-coding region, the boundary regions between the protein coding frames, and the 3' non-coding region. The locally optimal secondary structure occurring in the 5' terminal region has been assessed using different overlapping segment sizes and the Monte Carlo method. The results show that the most favorable structure for the 5' mRNA leader sequence of HIV has two stem-loops folded at nucleotides 5-104 in the R region (stem-loop I, 5-54 and stem-loop II, 58-104). A large fluctuation of segment score of the local optimal secondary structure also occurs in the boundary between the exterior glycosylated protein or outer membrane protein and transmembrane protein coding region. This finding is surprising since no RNA signals or RNA processing are expected to occur at this site. In addition, regions of the genome predicted to have significantly more open structure at the RNA level correlate closely with hypervariable sites found in these viral genomes. The possible importance of local secondary structure to the biological function of the human immunodeficiency virus genome is discussed.
Molecular cloning of biologically active proviruses of bovine immunodeficiency-like virus.
A series of independent proviral molecular clones of bovine immunodeficiency-like virus (BIV) obtained from a genomic library of BIV-infected bovine cell DNA were physically and biologically characterized. Heteroduplex mapping shows that two of these BIV clones (106 and 127) contain uninterrupted proviral sequences approximately 9.0 kb in length, flanked by nonhomologous bovine cellular sequences. Microinjection of purified DNA from BIV clone 106 or 127 into susceptible bovine cells produces virus-specific cytopathic effects, including syncytium induction, supernatant reverse transcriptase activity, and infectious virus particle formation, similar to the effects produced by parental virus stock. Using restriction enzyme mapping, it was determined that the two infectious clones share 13 of 14 sites mapped within the provirus; thus, based on this criterion, the two clones are nearly identical, with the exception of a single polymorphic site recognized in the 3' half of the genome. BIV appears to be an exogenous pathogenic virus, because Southern hybridization analyses detected no endogenous sequences related to BIV in DNA from a variety of uninfected bovine cells and tissues. Most of the BIV-related DNA found in cells 96 hr after infection is present as linear unintegrated viral DNA, although the presence of host flanking sequences in our proviral clones indicates that integration takes place. These biologically active clones of BIV will be of use in defining further the mechanisms of BIV pathogenesis and in engineering specific diagnostic reagents to determine the prevalence of BIV in cattle populations.
Sequence of MET protooncogene cDNA has features characteristic of the tyrosine kinase family of growth-factor receptors.
We isolated overlapping cDNA clones corresponding to the major MET protooncogene transcript. The cDNA nucleotide sequence contained an open reading frame of 1408 amino acids with features characteristic of the tyrosine kinase family of growth factor receptors. These features include a putative 24-amino acid signal peptide and a candidate, hybrophobic, membrane-spanning segment of 23 amino acids, which defines an extracellular domain of 926 amino acids that could serve as a ligand-binding domain. A putative intracellular domain 435 amino acids long shows high homology with the SRC family of tyrosine kinases and within the kinase domain is most homologous with the human insulin receptor (44%) and v-abl (41%). Despite these similarities, however, we found no apparent sequence homology to other growth factor receptors in the putative ligand-binding domain. We conclude from these results that the MET protooncogene is a cell-surface receptor for an as-yet-unknown ligand.
The visna virus genome: evidence for a hypervariable site in the env gene and sequence homology among lentivirus envelope proteins.
The complete nucleotide sequence of the visna virus 1514 genome was determined. Our sequence confirms the relationship of visna virus and other lentiviruses to human immunodeficiency virus (HIV) both at the level of sequence homology and of genomic organization. Sequence homology is shown to extend to the transmembrane proteins of lentivirus env genes; this homology is strongest in the extracellular domain, suggesting that close structural and functional similarities may also exist among these envelope proteins. Comparison of our data with the sequence of visna virus LV1-1, an antigenic variant derived from strain 1514, demonstrates that the rate of divergence has been about 1.7 x 10(-3) substitutions per nucleotide per year in vivo. This rate is orders of magnitude higher than that for most DNA genomes, but agrees well with estimates of the rate for HIV. A statistically significant cluster of mutations in the env gene appears to represent a hypervariable site and may correspond to the epitope responsible for the antigenic differences between 1514 and LV1-1. Analysis of the potential RNA folding pattern of the visna virus env gene shows that this hypervariable site falls within a region with little potential for intramolecular base pairing. This correlation of hypervariability with lack of RNA secondary structure is strengthened by the fact that it also holds for a hypervariable site in the env gene of HIV.
Structural and transcriptional analysis of human papillomavirus type 16 sequences in cervical carcinoma cell lines.
We cloned and analyzed the integrated human papillomavirus type 16 (HPV-16) genomes that are present in the human cervical carcinoma cell lines SiHa and CaSki. The single HPV-16 genome in the SiHa line was cloned as a 10-kilobase (kb) HindIII fragment. Integration of the HPV-16 genome occurred at bases 3132 and 3384 with disruption of the E2 and E4 open reading frames (ORFs). An additional 52-base-pair deletion of HPV-16 sequences fused the E2 and E4 ORFs. the 5' portion of the disrupted E2 ORF terminated immediately in the contiguous human right-flanking sequences. Heteroduplex analysis of this cloned integrated viral genome with the prototype HPV-16 DNA revealed no other deletions, insertions, or rearrangements. DNA sequence analysis of the E1 ORF, however, revealed the presence of an additional guanine at nucleotide 1138, resulting in the fusion of the E1a and E1b ORFs into a single E1 ORF. Sequence analysis of the human flanking sequences revealed one-half of an Alu sequence at the left junction and a sequence highly homologous to the human O repeat in the right-flanking region. Analysis of the three most abundant BamHI clones from the CaSki line showed that these consisted of full-length, 7.9-kb HPV-16 DNA; a 6.5-kb genome resulting from a 1.4-kb deletion of the long control region; and a 10.5-kb clone generated by a 2.6-kb tandem repeat of the 3' early region. These HPV-16 genomes were arranged in the host chromosomes as head-to-tail, tandemly repeated arrays. Transcription analysis revealed expression of the HPV-16 genome in each of these two cervical carcinoma cell lines, albeit at significantly different levels. Preliminary mapping of the viral RNA with subgenomic strand-specific probes indicated that viral transcription appeared to be derived primarily from the E6 and E7 ORFs.
Human T-cell lymphotropic virus type III shares sequence homology with a family of pathogenic lentiviruses.
The etiologic agent of the acquired immune deficiency syndrome, human T-cell lymphotropic virus type III (HTLV-III), has recently been shown to morphologically resemble and share sequence homology with visna virus, a pathogenic lentivirus. Molecular hybridization, heteroduplex mapping, and DNA sequence analyses were used to compare HTLV-III to other lentiviruses of domestic animals, including visna, caprine arthritis encephalitis, and equine infectious anemia viruses. Hybridization results showed that a substantial amount of sequence homology exists between each of these viruses and HTLV-III. In addition, a closer relationship was found between visna and caprine arthritis encephalitis viruses than for any of the other lentiviruses studied. These results, along with nucleotide and amino acid sequence comparisons, have been used in a comprehensive effort to derive a systematic relationship for lentiviruses and to provide further evidence for classifying HTLV-III with the Lentivirinae subfamily of retroviruses. This relationship predicts that similarities in biology and disease process can be expected between HTLV-III and other Lentivirinae members.
Nucleotide sequence of a transduced myc gene from a defective feline leukemia provirus.
The nucleotide sequence of a feline v-myc gene and feline leukemia virus (FeLV) flanking regions was determined. Both the nucleotide and predicted amino acid sequences are very similar to the murine and human c-myc genes (ca. 90% identity). The entire c-myc coding sequence is represented in feline v-myc and replaces portions of the gag and env genes and the entire pol gene. The coding sequence is in phase with the gag gene reading frame; v-myc, therefore, appears to be expressed as a gag-myc fusion protein. Viral sequences at the 3' myc-FeLV junction begin with the hexanucleotide CTCCTC, which is also found at the 3' fes-FeLV junction of both Gardner-Arnstein and Snyder-Theilen feline sarcoma viruses. These similarities suggest that some sequence specificity may exist for the transduction of cellular genes by FeLV. Feline v-myc lacks a potential phosphorylation site at amino acid 343 in the putative DNA-binding domain, whereas both human and murine c-myc have such sites. Avian v-myc has lost a potential phosphorylation site which is present in avian c-myc five amino acids from the potential mammalian site. If these sites are actually phosphorylated in normal c-myc proteins, their loss may alter the DNA-binding affinity of v-myc proteins.