The complete nucleotide sequence of cosmid vector pTL5: location and origin of its genetic components

The complete nucleotide sequence (5793 bp) of the cosmid vector pTL5 and the origin of its genetic components has been determined. Cosmid pTL5, a derivative of cosmid vector pHC79, is composed of genetic components from pBR322, bacteriophage [lambda] and the hybrid lambdoid bacteriophage Charon (Ch) 4A cohesive ends (cos) region. The Ch4A cos region contains genetic components from two bacteriophages, the [lambda] cos-left arm and the [phi]80 cos-right arm regions. The Ch4A cos region has been used in the construction of many other cosmid-type vectors, some of which have been sequenced and entered into the GenBank database.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31325/1/0000234.pd

Genotyping of Apolipoprotein E by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The genotyping of the various isoforms of Apolipoprotein E (apo E) has been performed using matrix-assisted laser desorption/ionization (MALDI-MS). The polymerase chain reaction was used to amplify the specific apo E gene sequence followed by digestion with Cfo I ( Clostridium formicoaceticum ), for generating restriction fragments for rapid and accurate mass analysis. An exonuclease I digestion step was introduced to remove the unused primers after PCR, which can otherwise interfere in the mass spectral analysis. By replacing the gel electrophoresis detection step with MALDI-MS, restriction isotyping of the apo E gene was achieved. Genotyping of an unknown sample obtained from an independent diagnostic laboratory demonstrated the validity of the MALDI-MS method for the routine clinical analysis of apo E. © 1998 John Wiley & Sons, Ltd.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/35073/1/281_ftp.pd

Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry as a Rapid Screening Method to Detect Mutations Causing Tay–Sachs Disease

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used as a rapid method for the detection of human genetic polymorphisms. In particular, the mutations in the human HEXA gene that cause the infantile Tay–Sachs disease have been studied using MALDI-MS to demonstrate the feasibility of this technique for use in clinical and diagnostic analysis. The protocols involved in this approach include, polymerase chain reaction for the amplification of the mutation site from buccal cell DNA, followed by restriction enzyme digestion of the amplified regions of the template cells. The products of amplification and digestion were studied using MALDI-MS. MALDI-MS experiments are shown to provide essentially the same information as obtained from gel electrophoresis but orders of magnitude faster. © 1997 John Wiley & Sons, Ltd.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/35066/1/981_ftp.pd

The in vivo endothelial cell translatome is highly heterogeneous across vascular beds

Endothelial cells (ECs) are highly specialized across vascular beds. However, given their interspersed anatomic distribution, comprehensive characterization of the molecular basis for this heterogeneity in vivo has been limited. By applying endothelial-specific translating ribosome affinity purification (EC-TRAP) combined with high-throughput RNA sequencing analysis, we identified pan EC-enriched genes and tissue-specific EC transcripts, which include both established markers and genes previously unappreciated for their presence in ECs. In addition, EC-TRAP limits changes in gene expression after EC isolation and in vitro expansion, as well as rapid vascular bed-specific shifts in EC gene expression profiles as a result of the enzymatic tissue dissociation required to generate single-cell suspensions for fluorescence-activated cell sorting or single-cell RNA sequencing analysis. Comparison of our EC-TRAP with published single-cell RNA sequencing data further demonstrates considerably greater sensitivity of EC-TRAP for the detection of low abundant transcripts. Application of EC-TRAP to examine the in vivo host response to lipopolysaccharide (LPS) revealed the induction of gene expression programs associated with a native defense response, with marked differences across vascular beds. Furthermore, comparative analysis of whole-tissue and TRAP-selected mRNAs identified LPS-induced differences that would not have been detected by whole-tissue analysis alone. Together, these data provide a resource for the analysis of EC-specific gene expression programs across heterogeneous vascular beds under both physiologic and pathologic conditions

Nucleotide Sequence Analysis of 77.7 kb of the Human V[beta] T-Cell Receptor Gene Locus: Direct Primer-Walking Using Cosmid Template DNAs

The nucleotide sequence of 77.7 kb from the human T-cell receptor [beta]-chain locus was determined directly from three overlapping cosmid clones using the primer-walking approach. Computer-aided analyses of this sequence reveal the presence of at least 11 genic regions that are closely related to the human T-cell receptor [beta] variable region (TCRBV) gene family. These include five germline sequences that have previously been determined, V[beta]21.2, V[beta]8.1, V[beta]8.2, V[beta]8.3, and V[beta]16, and four whose sequences have partially been determined at the mRNA level, V[beta]6, V[beta]23, V[beta]12.2, V[beta]24. The two remaining V[beta] Tcr-related sequences have eluded discovery by cDNA and RT-PCR cloning and genomic blot hybridization methods. These two V[beta] Tcr-related genes lack >75% nucleotide sequence identity with any other V[beta] Tcr gene member and therefore, by convention, are referred to as new subfamily members V[beta]25 and V[beta]26. This lack of shared identity with other subfamily members explains why they were not detected by hybridization. The promoter regions of these V[beta] Tcr genes contain the conserved Tcr decamer element located between 80 and 110 bp 5' of the translation start site, generally near a putative TATAA promoter element. Our sequence analysis also reveals that a 3.3-kb duplication unit was involved in the recombination event that produced the closely related V[beta]8.1 and 8.2 gene subfamily members. This sequenced region of the V[beta] locus contains an average number of repetitive DNA elements (21 Alu, three L1, three MER, and three retrovirus-related elements).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31701/1/0000637.pd

The in vivo endothelial cell translatome is highly heterogeneous across vascular beds

Endothelial cells (ECs) are highly specialized across vascular beds. However, given their interspersed anatomic distribution, comprehensive characterization of the molecular basis for this heterogeneity in vivo has been limited. By applying endothelial-specific translating ribosome affinity purification (EC-TRAP) combined with high-throughput RNA sequencing analysis, we identified pan EC-enriched genes and tissue-specific EC transcripts, which include both established markers and genes previously unappreciated for their presence in ECs. In addition, EC-TRAP limits changes in gene expression after EC isolation and in vitro expansion, as well as rapid vascular bed-specific shifts in EC gene expression profiles as a result of the enzymatic tissue dissociation required to generate single-cell suspensions for fluorescence-activated cell sorting or single-cell RNA sequencing analysis. Comparison of our EC-TRAP with published single-cell RNA sequencing data further demonstrates considerably greater sensitivity of EC-TRAP for the detection of low abundant transcripts. Application of EC-TRAP to examine the in vivo host response to lipopolysaccharide (LPS) revealed the induction of gene expression programs associated with a native defense response, with marked differences across vascular beds. Furthermore, comparative analysis of whole-tissue and TRAP-selected mRNAs identified LPS-induced differences that would not have been detected by whole-tissue analysis alone. Together, these data provide a resource for the analysis of EC-specific gene expression programs across heterogeneous vascular beds under both physiologic and pathologic conditions

Whole exome sequencing of ENU-induced thrombosis modifier mutations in the mouse.

Although the Factor V Leiden (FVL) gene variant is the most prevalent genetic risk factor for venous thrombosis, only 10% of FVL carriers will experience such an event in their lifetime. To identify potential FVL modifier genes contributing to this incomplete penetrance, we took advantage of a perinatal synthetic lethal thrombosis phenotype in mice homozygous for FVL (F5L/L) and haploinsufficient for tissue factor pathway inhibitor (Tfpi+/-) to perform a sensitized dominant ENU mutagenesis screen. Linkage analysis conducted in the 3 largest pedigrees generated from the surviving F5L/L Tfpi+/- mice ('rescues') using ENU-induced coding variants as genetic markers was unsuccessful in identifying major suppressor loci. Whole exome sequencing was applied to DNA from 107 rescue mice to identify candidate genes enriched for ENU mutations. A total of 3,481 potentially deleterious candidate ENU variants were identified in 2,984 genes. After correcting for gene size and multiple testing, Arl6ip5 was identified as the most enriched gene, though not reaching genome-wide significance. Evaluation of CRISPR/Cas9 induced loss of function in the top 6 genes failed to demonstrate a clear rescue phenotype. However, a maternally inherited (not ENU-induced) de novo mutation (Plcb4R335Q) exhibited significant co-segregation with the rescue phenotype (p = 0.003) in the corresponding pedigree. Thrombosis suppression by heterozygous Plcb4 loss of function was confirmed through analysis of an independent, CRISPR/Cas9-induced Plcb4 mutation (p = 0.01)