Biomedical subjects
Publications and source records attributed to J Brosius.
Using a simple electronic circuit, a thermocouple can be connected to a chart recorder to measure the actual temperature inside a PCR tube. This allows accurate inspection of the thermocycle program and comparison between thermoprofiles from different thermocyclers. We found that the recording of temperature cycling enabled us to obtain more reliable and reproducible results.
Versatile DNA polylinkers of more than 300 bp were constructed. They contain the recognition sequences of all restriction enzymes--whether known or still to be discovered--that recognize palindromic hexamers. In addition to these 64 uninterrupted hexameric recognition sites, a number of sites containing interrupted palindromes and nonpalindromic sequences and two recognition sequences with 8 bp are present. Polylinkers (in several variants) were inserted into frequently utilized Escherichia coli cloning vectors such as pBluescript (yielding pSLJ10, pSL250, pSL260, pSL270, and pSL300), pUC18/pUC19 (yielding pSL180 and pSL190, respectively), or pUC118/pUC119 (yielding pSL1180 and pSL1190, respectively). A subtle color discrimination between presence and absence of insert in pSL300 (mid-blue to light-blue or white) was seen in a number of test ligations. The mid-blue color that is generated by pSL300 is presumably due to translational restarts. A different intergenic region for translational restarts was used in plasmids pSL251, pSL261, pSL271, and pSL301. The polylinker was also inserted into expression vector pUC120, yielding pSE1200, and into expression vector pKK233-2, yielding pSE220 and a shortened version thereof, pSE280. Finally, the polylinker was inserted into pTrc99A, resulting in pSE380, which carries a lac repressor gene. This expands the use of the expression system beyond lacIq strains to other bacterial hosts. These versatile vectors have broad applications in genetic engineering.
Explore the source record for details and available documents.
Diazepam binding inhibitor (DBI), an endogenous 10-kDa polypeptide was isolated from rat and human brain by monitoring displacement of radioactive diazepam bound to specific recognition sites in brain synaptic and mitochondrial membranes. The cellular location of DBI mRNA was studied in rat brain and selected peripheral tissues by in situ hybridization histochemistry with a 35S-labeled single-stranded complementary RNA probe. DBI mRNA was heterogeneously distributed in rat brain, with particularly high levels in the area postrema, the cerebellar cortex, and ependyma of the third ventricle. Intermediate levels were found in the olfactory bulb, pontine nuclei, inferior colliculi, arcuate nucleus, and pineal gland. Relatively low but significant levels of silver grains were observed overlying many mesencephalic and telencephalic areas that have previously been shown to contain numerous DBI-immunoreactive neurons and a high density of central benzodiazepine receptors. In situ hybridizations also revealed high levels of DBI mRNA in the posterior lobe of the pituitary gland, liver, and germinal center of the white pulp of spleen, all tissues that are rich in peripheral benzodiazepine binding sites. The tissue-specific pattern of DBI gene expression described here could be exploited to further understand the physiological function of DBI in the brain and periphery.
AP50 is the 50,000-dalton protein component found in clathrin-coated vesicles as part of the coat assembly protein (AP) complex, AP-2. AP50 cDNA clones were isolated from rat brain cDNA libraries, and their nucleotide sequence was determined. The isolated cDNA clones represent the entire coding sequence for the rat brain AP50. They encode a polypeptide containing 435 amino acids with a molecular weight of 49,612 daltons. Comparison with the partially sequenced bovine brain AP50 shows a primary structure that is highly conserved. AP50 does not have detectable sequence similarity with other known kinases or with other proteins of known sequence.
The clathrin light chains fall into two major classes, LCA and LCB. In an intact clathrin triskelion, one light chain, of either class, is bound to the proximal segment of a heavy chain leg. Analysis of rat brain and liver complementary DNA clones for LCA and LCB shows that the two light chain classes are closely related. There appear to be several members of each class having deletions of varying length aligned at the same position. A set of ten heptad elements, characteristic of alpha-helical coiled coils, is a striking feature of the central part of each derived amino acid sequence. These observations suggest a model in which the alpha-helical segment mediates binding to clathrin heavy chains and the amino- and carboxyl-terminal segments mediate interactions with other proteins. They also suggest an explanation for the observed tissue-dependent size variation for members of each class.
Explore the source record for details and available documents.
We have deduced the 1675-amino acid sequence of rat clathrin heavy chain from cDNA clones and predict a protein of Mr 191,569. We have established the polarity of the heavy chain and assigned sequence positions to several structural landmarks of the clathrin leg. The terminal domain at the distal end of the clathrin leg is at the amino terminus of the heavy chain. It is connected to the distal segment by a flexible "link" from Tyr-479 to Arg-523. There is an unusual sequence at the carboxyl terminus that may form the globular projection at the vertex of the clathrin trimer. We suggest that a possible site of heavy-chain-light-chain interaction is located in the proximal segment. Comparison with other partially sequenced mammalian clathrin heavy chains shows that the primary structure is highly conserved. The heavy chain is unrelated to other classes of structural proteins.
BC1 is a small developmentally regulated RNA that is prevalent in nervous tissue. In order to determine if BC1 RNA represents the transcriptional by-product of various repetitive brain identifier (ID) elements or the independent transcript of a single or a few genes, we compared the sequences of a population of cDNA clones derived from in vitro C-tailed BC1 RNA. Each of 10 randomly selected clones revealed a 5' domain that was identical in sequence to the ID element, followed by an internal region of poly(A). In 8 of the clones, we found an identical, nonrepetitive sequence domain located at the 3' end of each molecule. An oligonucleotide of 30 residues complementary to this section identified only BC1 RNA in blot-hybridization analysis. Our results strongly suggest that BC1 RNA is transcribed specifically from the BC1 gene(s) and is not a highly heterogeneous population of ID-containing RNA polymerase III transcripts. Moreover, the availability of a unique BC1 RNA sequence will facilitate studies on tissue- and stage-specific gene regulation and will help in clarifying the role of this small RNA in the brain.
We report the isolation of a clone encoding calmodulin from a rat brain cDNA library; genomic clones were also isolated and partially characterized. The derived amino acid sequence has 100% homology to the published mammalian protein sequences. In rat, we identify two major RNA species of approximately 2.2 and 0.75 kb in length by RNA blot analysis. Furthermore, the mRNA is highly enriched in brain tissue, compared to liver or kidney. A variant (GATAAA) of the putative poly(A) addition signal is observed at the 3' end of one of the mRNA species.
cDNA clones corresponding to the polypeptide that has been shown to be an endogenous diazepam binding inhibitor and may act as a physiological ligand for the benzodiazepine/beta-carboline receptor have been isolated from bacteriophage lambda recombinant libraries from rat hypothalamus, total brain, and liver. The clones contain an open reading frame corresponding to 87 amino acids. A signal sequence is not present. In addition to high levels of mRNA in various brain regions, RNA blot analysis reveals an abundance of diazepam binding inhibitor mRNA in many peripheral organs (e.g., testes, kidney, liver, and heart) that are known to be rich in peripheral benzodiazepine recognition sites. The size of the mRNA in all tissue examined is approximately 0.7 kilobase. Southern blot analysis of genomic DNA suggests the presence of about six genes in the rat, some of which may be pseudogenes.
We have isolated and characterized DNA segments containing IFN-alpha-related sequences from human lambda and cosmid clone banks. We describe six linkage groups comprising 18 distinct IFN-alpha-related loci, and report the nucleotide sequences of nine chromosomal IFN-alpha-genes with intact reading frames, as well as of five pseudogenes. Taking into account as yet unsequenced genes as well as clones described by others, there are now seven linkage groups and 23 loci, of which 15 correspond to potentially functional genes and six to non-functional genes; two loci remain unsequenced. Eighteen additional sequences are likely to be allelic to the above. The finding that at least two IFN-alpha genes appear to be natural hybrids of other IFN-alpha genes, and that two distinct IFN-alpha loci have completely identical coding sequences, although their flanking regions are different, is evidence for information exchange between the individual genes.
In the tac promoter (deBoer, H. A., Comstock, L. J., and Vasser, M. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 21-25) the spacing between the -35 and -10 consensus sequences is 16 base pairs. Between these two regions we inserted 1 or 2 base pairs to increase the distance to 17 base pairs (trc promoter) or 18 base pairs (tic promoter). The activities of the three promoters were compared in vivo by fusion to the chloramphenicol acetyltransferase or to the Escherichia coli 4.5 S RNA gene. Both measurements gave consistent results. The trc and tic promoters are on average about 90% and 65% as active as the tac promoter, respectively.
We describe the characterization in vitro of three hybrid trp-lacUV5 (TAC) promoters, which have perfect homology to the -10 and -35 Escherichia coli promoter consensus hexamer sequences, but which differ in the distance between the -10 and -35 regions. The three promoters, TAC16, TAC17, and TAC18 have spacings of 16, 17, and 18 base pairs, respectively. We have measured KB and k2, the constants that describe the formation of open complexes, on these three promoters. We have also measured their relative strengths on supercoiled plasmid DNA. Our results show that these are the strongest promoters that have been characterized in vitro so far and confirm the hypothesis that the consensus promoter sequence is "best." We find the TAC17 promoter (KBk2 = 8.4 X 10(7) M-1 s-1) to be stronger than either the TAC16 (KBk2 approximately 1.5 X 10(7) M-1 s-1) or TAC18 (KBk2 approximately 3.5 X 10(7) M-1 s-1) promoters, a result that is in agreement with other findings on the effect of spacer length. The choice of start point for transcription is affected by spacer length. Transcription from all the promoters was stimulated at moderate concentrations of salt (less than 150 mM) and persisted at high salt concentrations (300 mM).
In order to characterize the tandem rrnB promoters transcribing one of the ribosomal RNA operons in E. coli we subcloned the basic promoter unit. This 185 bp fragment extends from -64 to +121 counted from the transcription start site of upstream promoter P1. The start site of downstream promoter P2 is also included in the promoter cartridge. S1 mapping experiments show that both promoters on this fragment are active in vivo. BAL-31 deletion mutations generated at the start site for promoter P2 were also tested by S1 mapping. Transcription from P2 remained active in all cases with the exception of one construction which lacks the -10 region. This demonstrates that the sequences downstream from the -10 region of P2 are not essential for basic promoter function.
A plasmid cloning vector system has been constructed that allows for the production of large quantities of foreign proteins or fragments thereof, in an unfused state. These vectors provide strong regulated trp-lac fusion promoters and the lacZ ribosome-binding site (RBS) followed by an ATG translation initiation codon at an appropriate distance from the RBS. The ATG codon is located within a unique NcoI restriction site (CCATGG). Digestion with NcoI exposes the ATG for fusion. Gene fragments lacking a prokaryotic RBS and/or ATG start codons can be inserted in several ways. Expression experiments using a truncated cI gene of bacteriophage lambda or a large portion of the coding region of the Herpes simplex virus type 1 glycoprotein D gene have been performed. The results of these studies show that the vectors are useful for the high-level expression of prokaryotic and eukaryotic genes in Escherichia coli.
Vectors were constructed which contain promoterless genes for chloramphenicol (cam) or tetracycline (tet) resistance, as promoter-probe plasmids. Escherichia coli cells harboring these plasmids are sensitive to cam or tet but resistant to ampicillin. In plasmids pKK231 -1 and pKK232 -8 the gene for cam acetyltransferase (CAT) and in pKK175 -6 the gene for tet resistance are flanked by efficient transcription terminators, preventing transcription from other pBR322 promoters into the antibiotic resistance region. In one of the vectors, pKK232 -8, translational stop codons were introduced in all three reading frames upstream from the initiation codon of the cat gene. If a DNA fragment containing a promoter is inserted into one of the cloning sites upstream from the antibiotic genes, cells carrying such plasmids acquire resistance to cam or tet. Using these vectors two restriction fragments that contain promoters were identified. One of these fragments contains sequences upstream from an unidentified gene ( ORFII ) located distal to the rrnB rRNA operon of E. coli.
A rat insulin gene which was fused to Escherichia coli signals for the initiation of translation could not be retained when expressed from the strong rrnB ribosomal RNA promoters or an induced trp/lac (= tac) hybrid promoter. When the latter promoter was repressed by transformation into a lac-repressor-overproducing strain, the insulin gene fragment could be retained. Upon induction of the promoter with isopropyl-beta-D-galactosidase the growth rate of the cells was reduced, and in most cases the cells subsequently lysed. Deletion of the translational initiation signals, changing the reading frame, or insertion of an efficient transcription terminator between the promoter and the rat insulin gene each permitted retention of the fragment. The first two observations indicate that overproduction of the specific polypeptide, and not of the RNA, is detrimental to the cell. The third finding has been exploited for the testing and selection of transcription terminators. The rpoC terminator, which is located distal to the rplJL /rpoBC operon, has been shown to terminate transcripts from the rrnB promoters. It was also shown that the putative rrnB terminators, T1 and T2, each function separately in vivo.