A lambda insertion type cDNA cloning vector, Lambda ZAP, has been constructed.In E. coli a phagemid, pBluescnpt SK(-), contained within the can be excised by f1 or M13 helper phage.The excision process eliminates need to subclone DNA inserts from phage into plasmid restrction digestion and ligation.This is possible because ZAP incorporates signals for both initiation termination of synthesis fI bacteriophage origin replication (1).Six 21 restriction sites in pBluescript SK polyflinker,...

Transgenic mice with a lambda shuttle vector containing lacI target gene were generated for use as short-term, in vivo mutagenesis assay. The is recovered from the treated by exposing mouse genomic DNA to vitro packaging extracts and plating rescued phage on agar plates 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-Gal). Phage mutations form blue plaques, whereas nonmutated colorless plaques. Spontaneous background mutant rates using this system range 0.6 x 10(-5) 1.7 10(-5),...

Mutations in the human glucocerebrosidase gene cause Gaucher disease. A cDNA clone containing entire coding region from normal cells has been isolated using lambda gt11 expression libraries. The complete nucleotide sequence, a restriction map, and hydropathy profile are presented. Hybridization to chromosome-specific DNA localizes chromosome 1. likely precursor protein is 515 amino acids long. NH2-terminal 19 constitute leader sequence that cleaved mature protein. predicted molecular weight...

Efficient generation of catalytic antibodies is uniquely dependent on the exact nature binding interactions in antigen-antibody complex. Current methods for monoclonal do not efficiently survey immunological repertoire and, therefore, they limit number catalysts that can be obtained. We are exploring to clone and express Escherichia coli. As essential first step, we present here a method establishment highly diverse heavy chain variable region library. Consequently, it should now possible...

A short term, in vivo mutagenesis assay has been developed utilizing a lacl target gene contained within lambda ZAP shuttle vector which incorporated into transgenic mice. Following chemical exposure, the was recovered from mouse genomic DNA by mixing with vitro phage packaging extract. Mutations were identified infecting host E. coli packaged and plating on indicator plates containing Xgal. Phage plaques mutations appeared blue, while intact colorless. The ratio of blue to colorless is...

The full-length gene that encodes the light chain variable regions of an idiotypically related group human IgM kappa rheumatoid factors (RFs) has been cloned and sequenced. deduced amino acid sequence is identical to four separate RF proteins. These results prove genes capable encoding anti-IgG autoantibody chains without any somatic mutation are present in repertoire normal people.

Amphotropic retroviral expression systems were used to synthesize hepatitis B virus surface antigen (HBsAg) and core antigen. The vectors permitted establishment of cell lines which expressed from either the long terminal repeat or mouse metallothionein-I promoter. HBsAgs synthesized containing no pre-S sequences, pre-S(2) sequences alone, pre-S(1) plus sequences. Inclusion did not affect secretion density HBsAg particles but reduce their mass by approximately 30%. Addition almost completely...

Transgenic mouse lineages were established that carry the normal (M) or mutant (Z) alleles of human α 1 -antitrypsin (α -Pi) gene. All -Pi transgenic mice expressed protein in liver, cartilage, gut, kidneys, lymphoid macrophages, and thymus. The M-allele was secreted normally into serum. However, Z-allele accumulated several cell types, but particularly hepatocytes, found serum tenfold lower concentrations than protein. Mice one lineage carrying Z allele high levels RNA displayed significant...

Gaucher disease is an autosomal recessive glycolipid storage characterized by a deficiency of glucocerebrosidase. The most common in persons Ashkenazi Jewish ancestry and the mutation, accounting for about 75% mutant alleles this population, known to be A----G substitution at cDNA nucleotide (nt) 1226. Screening has not been possible because nearly 25% had identified, but linkage analysis led suggestion that these could accounted single mutation. We now report discovery insertion nucleotide,...

The molecular diagnosis of Gaucher disease has been difficult due to the existence several different point mutations in glucocerebrosidase gene and presence a tightly linked, highly homologous pseudogene. We now report occurrence "Lepore-like" fusion which 5' end is functional 3' This further complicates but sheds light on anatomy complex pathogenesis this important storage disease.

Transgenic mice suitable for the in vivo assay of uspected mutagens at chromosome level have been constructed by stable integration a lambda phage shuttle vector. The vector, which contains /S-galactosidase (/3-gal) target gene, can be rescued from genomic DNA with vitro packaging extracts. Mutations In gene are detected change plaque color on indicator agar plates. Initial rescue efficiencies less than 1 forming unit (pfu)/100/ig were too low mutation analysis. We determined cause examining...

Retroviral vectors have been constructed for gene transfer in mammalian and avian cells, however most retroviral vector systems are complicated by the spread of a replication-competent helper virus. This problem has circumvented segregating viral genome into cis- trans-acting components. By establishing cell lines that produce products, one can propagate cis-acting component them harvest defective particles contain only component. The provide useful vehicle highly efficient genes target...

The v-src gene of Schmidt-Ruppin strain A Rous sarcoma virus is flanked by a 115-nucleotide direct repeat. Mutants that lack either the upstream or downstream copy replicate normally. However, mutants both copies do not replicate. Cloned viral DNA lacking sequence capable directing transcription RNA posttransfection. This polyadenylated, spliced, exported from nucleus, and translated into protein virions isolated culture medium 48 h posttransfection RNA. When mutant contransfected with...

Glucocerebrosidase cDNA and the neomycin-resistance gene (neo) were cloned into a retrovirus vector. Mouse fibroblasts infected with this vector expressed human glucocerebrosidase, which was readily distinguished from mouse enzyme using monoclonal anti-glucocerebrosidase antibodies. Cultured transformed lymphoblasts patients type I Gaucher disease rescued by helper virus. Transformed cells selected antibiotic G418. The activity of virus containing glucocerebrosidase restored to normal, while...

Z mutant-associated alpha 1-antitrypsin deficiency in human beings leads to hepatitis and, some cases, hepatocellular carcinoma. To begin delineate the molecular basis for development of carcinoma deficiency, we previously developed transgenic mice using M and genomic clones. High-copy lineage (12 gene copies/haploid mouse genome; "Z#2") had hepatocytes distended with globules. Hepatitis was present, morphological changes mimicked those observed deficiency-related liver disease. The numbers...

Gaucher disease is due to mutations involving the glucocerebrosidase gene. A closely homologous pseudogene located approximately 16 kD downstream from functional Sequence analysis of clones cDNA libraries made skin fibroblast cultures showed several independent with sequence an aberrantly processed message. Examination cellular RNA lymphoblasts or fibroblasts obtained thirteen patients, one heterozygote, and four normal subjects that was consistently transcribed, in some cases level...

Membrane glycoprotein (GP) IIb-IIIa is a component of platelet adhesive protein receptor. A region the heavy chain GPIIb, defined by monoclonal antibody PMI-1, involved in adhesion receptor function. We have localized and chemically synthesized this GPIIb. cDNA clone that directs synthesis fusion reactive with PMI-1 was isolated from phage lambda gt11 expression library constructed mRNA an erythroleukemia (HEL) cell line. The deduced amino acid sequence indicates it spans light-heavy...