Biomedical subjects
S M Weissman
Publications and source records attributed to S M Weissman.
Construction of lambda gt103, a derivative of phage lambda gt10 that has unique EcoRI, NotI, SacI and SpeI sites and retains positive selection for recombinants.
A phage vector, lambda gt103, that has unique EcoRI, NotI, SacI and SpeI sites within the imm434 cI repressor gene, was constructed by PCR-aided site-directed mutagenesis of lambda gt10 [Huynh et al., DNA Cloning Techniques: A Practical Approach, 1985, pp. 49-78]. This vector allows directional cloning and retains positive selection for recombinants on Escherichia coli C600hfl strains (since only phages with disrupted cI genes plate on this host). Libraries made with this phage vector can be efficiently screened for clones in which a part of the insert is homologous to probe DNAs derived from a plasmid-based library, without cross-hybridization.
Characterization of cDNA for the large subunit of the transcription initiation factor TFIIF.
At least six chromatographically resolvable general transcription factors may participate in accurate initiation by RNA polymerase II in HeLa cell-derived systems. TFIIF (also termed FC, RAP30/74 and beta/gamma) can bind directly to RNA polymerase II in solution and decrease the affinity of RNA polymerase II for nonspecific DNA. From studies on the kinetics of transcription initiation, on the composition of transcription initiation complexes fractionated by acrylamide gel electrophoresis, and on template competition experiments, TFIIF is known to act at an intermediate stage in initiation complex formation. It acts after TFIID firmly associates with DNA, but coincidentally with or immediately after RNA polymerase II binding to DNA, and before the recruitment of factor TFIIE. TFIIF may or may not have DNA helicase activity. The small subunit (RAP30) of TFIIF has been cloned and shows some amino-acid sequence homology to bacterial sigma factors. We have partially sequenced the RAP74 protein from purified HeLa cells, cloned its complementary DNA and shown that its translation product can interact with RAP30 in vitro as well as in vivo. The cDNA predicts an amino-acid sequence that lacks obvious DNA or RNA helicase motifs. It has regions rich in charged amino acids, including segments containing a higher content of acidic amino acids than are found in strong transcriptional activators such as VP16.
Chromosome fishing: an affinity capture method for selective enrichment of large genomic DNA fragments.
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Physical and genetic mapping of the telomeric major histocompatibility complex region in man and relevance to the primary hemochromatosis gene (HFE).
We performed pulsed-field gel electrophoresis (PFGE) on genomic DNA from a radiation hybrid (RH) cell line and constructed a high-resolution physical map of the major histocompatibility complex class I region in 6p21.3, where the gene for primary hemochromatosis (HFE) is believed to be located. Due to the intact microsegment of hemizygous human genomic DNA preserved in the RH cell line, simplified and distinct restriction fragment banding patterns were generated. Using the RH cell line, we were able to extend the physical map of the HLA class I region to about 3000 kb, order the known HLA class I genes from centromere to telomere: HLA-B, -C, -E, (-A, -H, -G), and -F, and orient the HLA-F gene along the chromosome. The proximity of HLA-F to HLA-A was confirmed by linkage and linkage disequilibrium analysis. This study shows that RH cell lines can be useful for constructing long-range physical maps in specific regions of the human genome with PFGE. Physical and genetic mapping studies of this region are consistent with a localization of the HFE gene proximal or distal to HLA-A.
The core protein of epican, a heparan sulfate proteoglycan on keratinocytes, is an alternative form of CD44.
Epican, a heparan sulfate proteoglycan, was recently identified on the surface of keratinocytes with the aid of a monoclonal antibody to its core protein. Using that antibody to screen a human keratinocyte cDNA library, a clone encoding the entire epican core protein was selected and sequenced. The core protein of epican is a form of CD44. The deduced protein sequence of 699 amino acids has a novel 339 amino acid domain inserted into the proximal extracellular domain of the standard, leukocyte form of CD44. The additional domain adds a number of potential N- and O-linked glycosylation sites and two proteolysis sites to this form of CD44.
Occupancy of upstream regulatory sites in vivo coincides with major histocompatibility complex class I gene expression in mouse tissues.
The major histocompatibility complex (MHC) class I HLA-B7 transgene carrying a 660-bp upstream sequence is expressed in the mouse with tissue specificity that parallels that of the expression of endogenous mouse MHC class I (H-2) genes. We have performed in vivo genomic footprinting for the HLA-B7 transgene and the endogenous H-2Kb gene. We show that the upstream region of both the transgene and the endogenous gene was extensively occupied in spleen tissue, where these genes are expressed at high levels. In contrast, no occupancy was detected in brain tissue, where expression of these genes is virtually absent. Sites exhibiting in vivo protection correspond to cis elements previously shown to bind to nuclear factors in vitro, including the constitutive enhancer region I and the interferon response element. The strongest tissue-specific protection was detected at site alpha, located downstream from the interferon response element. Site alpha bound a constitutively expressed nuclear factor(s) in vitro that exhibited an overlapping specificity which may involve a nuclear hormone receptor, RXR, and an AP-1-related factor. Site alpha was functional in vivo, as it enhanced MHC class I transcription in lymphocytes. These results show that the tissue-specific occupancy of the MHC class I regulatory sequences in vivo correlates with their expression and suggest that in vivo occupancy is controlled by a mechanism other than the mere presence of factors capable of binding to these sites. Our results suggest that a sequence present in the 660-bp upstream region in a human leukocyte antigen gene directs tissue-specific occupancy of MHC class I genes in vivo, independently of their position and copy number, illustrating a potential advantage of using a transgene for delimitation of the sequence requirement for in vivo occupancy.
A contingent replication assay for the detection of protein-protein interactions in animal cells.
We have developed a sensitive and rapid assay system termed the contingent replication assay (CRA) for selecting cDNAs with desired functional properties from a cDNA library. The system functions in animal cells and permits enrichment of the desired cDNA in small-scale and convenient experiments. The assay can be used for the enrichment of proteins that activate transcription from conditional enhancers, bind to specific DNA sequences, or interact with target proteins of interest. In this communication we report the application of this assay to study protein-protein interactions in animal cells.
cDNA selection: efficient PCR approach for the selection of cDNAs encoded in large chromosomal DNA fragments.
Identification of coding segments in large fragments of genomic DNA is a recurrent problem in genome mapping and positional cloning studies. We have developed a rapid and efficient protocol to achieve this goal, based on hybridization of cDNA fragments to immobilized DNA and recovery of the selected cDNAs by the PCR. The procedure permits rapid cloning of cDNA fragments encoded by large genomic DNA fragments, groups of yeast artificial chromosomes, or cosmids and has the potential to directly enrich cDNAs encoded in chromosome segments. By this approach we have been able to identify several non-major histocompatibility complex class I clones from a yeast artificial chromosome that includes the HLA-A locus.
Differential expression of novel Gs alpha signal transduction protein cDNA species.
The Gs alpha guanine nucleotide-binding signal transduction protein is part of a heterotrimeric complex that is involved in the stimulation of adenylate cyclase upon activation of membrane receptors. We report the characterization of 16 Gs alpha cDNA clones isolated from the human adult retina and fetal eye libraries. Molecular heterogeneity in the 5'-region defines four novel Gs alpha cDNA species which are generated either by alternate splicing or by using alternative promoter. The novel exons upstream of exon 2 interrupt the highly conserved 'region A' in the Gs alpha polypeptide. Non-AUG codons in the novel 5'-exon can initiate translation of these Gs alpha species in vitro. Reverse transcription of total RNA coupled with polymerase chain reaction (RTPCR) using specific primers and in situ hybridization to mRNA in baboon tissue sections with a specific oligonucleotide probe show a high level of expression of these species in retina and brain but not in liver. Differential expression of alternatively spliced Gs alpha species suggests novel signal transducing pathways.
Purification of BsuE methyltransferase and its application in genome mapping.
We have used a combination of BsuE methyltransferase (M-BsuE) and NotI restriction enzyme to cut genomic DNA at a subset of NotI sites. The usefulness of this system is shown in a re-examination of the restriction map of the human MHC. Combinations of methylases and restriction enzymes can be used to generate cuts at different frequencies in genomic DNA, such that they generate ends complementary to NotI ends, and can be used in conjunction with NotI linking clones in chromosome jumping experiments. These enzyme combinations have the potential to produce cutting sites in genomic DNA spaced at intervals favorable for extensive mapping, fragment enrichment, and cloning efforts.
Isolation and molecular characterization of a cDNA encoding the 23-kDa subunit of human RNA polymerase II.
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A simple and efficient cDNA library subtraction procedure: isolation of human retina-specific cDNA clones.
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Construction of a uniform-abundance (normalized) cDNA library.
We have used a kinetic approach to construct cDNA libraries containing approximately equal representations of all sequences in a preparation of poly(A)+ RNA. Randomly primed cDNA fragments of a selected size range were cloned in lambda phage vector. Inserts were amplified by the polymerase chain reaction (PCR), denatured, and self-annealed under optimized conditions. After extensive but incomplete reannealing, the single-stranded fraction was relatively depleted of more abundant species of cDNA. Libraries of these fragments are suitable for cDNA subtraction, screening, or selection by hybridization and make it possible to detect and analyze cDNA corresponding to species of mRNA present at a low level in a small fraction of the cells in a complex tissue.
Construction and characterization of a NotI-BsuE linking library from the human X chromosome.
We describe the construction and characterization of methylation-resistant sequence-tagged NotI linking clones specific for the X chromosome, referred to as NotI-BsuE linking clones. The approach consists of methylating the X-chromosome-specific cloned DNA with BsuE methylase (M. BsuE), an enzyme that methylates the first C residue in the CGCG sequence, followed by selection of the methylation-resistant NotI sites by insertion of a kanamycin-resistance gene in the clones cleavable by NotI. The frequent occurrence of NotI sites in CpG islands is expected to cause methylation of a large number of NotI sites with BsuE methylase, thereby rendering them resistant to NotI cleavage. Thus, the combination of M. BsuE and NotI yields less frequent cutting than the NotI alone. We have isolated, partially sequenced, and characterized 113 NotI-BsuE linking clones, and mapped 50 clones to various regions along the chromosome.
Long-term soft tissue response to LeFort I maxillary superior repositioning.
The purpose of this study was to compare short-term (approximately 12 months postoperatively) and long-term (24 to 81 months postoperatively) records of 36 patients who underwent superior repositioning of the maxilla via LeFort 1 osteotomies. The parameters of measurement were set up to analyze the net response of the soft tissues of the face to superior repositioning of the maxilla. The results of this study show that soft-tissue changes associated with maxillary impaction are minimal and that no significant differences exist between twelve-month records and five-year records.
Identification of cis sequences controlling efficient position-independent tissue-specific expression of human major histocompatibility complex class I genes in transgenic mice.
We previously reported that genomic major histocompatibility complex class I human leukocyte antigen (HLA)-B7 gene constructs with as little as 0.66 kb of 5'- and 2.0 kb of 3'-flanking DNA were expressed efficiently and appropriately in transgenic mice. To identify and characterize the relevant cis-acting regulatory elements in more detail, we have generated and analyzed a series of transgenic mice carrying native HLA-B7 genes with further 5' truncations or intronic deletions and hybrid constructs linking the 5'-flanking region of B7 to a reporter gene. We were unable to detect a specific requirement for sequence information within introns 2 to 7 for either appropriate constitutive or inducible class I expression in adult animals. The results revealed the presence of cis-acting regulatory sequences between -0.075 kb and -0.66 kb involved in driving efficient copy number-dependent constitutive and gamma interferon-enhanced tissue-specific expression. The region from -0.11 to -0.66 kb is also sufficient to prevent integration site-specific "position effects," because in its absence HLA-B7 expression is frequently detected at significant levels at inappropriate sites. Conserved sequence elements homologous to the H-2 class I regulatory element, or enhancer A, and the interferon response sequence are located between about -151 and -228 bp of the B7 gene. Our results also indicate the existence of sequences downstream of -0.11 kb which can influence the pattern of tissue-specific expression of the HLA-B7 gene and the ability of this gene to respond to gamma interferon.
Incorporation of facial plastic surgery in the planning and treatment of orthognathic surgical cases.
Until recently, orthognathic surgery and cosmetic soft tissue surgery have been separate clinical endeavors. This article presents examples of soft tissue surgical procedures carried out with orthognathic surgery to enhance the overall result. Video imaging techniques have been integrated into the planning process to more clearly define functional and esthetic goals. The technical achievement of combined orthognathic and esthetic procedures, as well as the timing of treatment between the orthodontist, oral surgeon, and plastic surgeon, is discussed.