J H Weis

Expression of hemolytically active murine fourth component of complement in transfected L cells

The S region of the murine major histocompatibility complex contains two related genes (C4-X and C4-Y), one of which encodes the fourth component of complement (C4). We have introduced cloned C4-X and C4-Y genes into L cells. C4-X transfectants contained no detectable C4-related RNA, whereas C4-Y transfectants contained large amounts of C4-related RNA. Culture supernatants of C4-Y transfectants contained processed C4 which was indistinguishable from macrophage-derived C4. The L-cell-derived C4 restored hemolytic activity to C4-deficient serum, served as a substrate for C1s, and underwent autolytic cleavage suggesting the presence of an intramolecular thiolester bond. These experiments identify C4-Y as the functional C4 gene in H-2d mice and indicate that L cells can process pro-C4 correctly.

Eucaryotic chromosome transfer: production of a murine-specific cosmid library from a neor-linked fragment of murine chromosome 17.

We recently developed a procedure for the molecular analysis of specific mammalian chromosomal fragments. This procedure allows for the transfer of contiguous chromosomal fragments, varying in size from a fraction to several centimorgans in length, from the donor cell of one species into a recipient cell of a different species. Specifically, we inserted a neor gene, encoded by a recombinant retrovirus, into the murine major histocompatibility complex (MHC). Metaphase chromosome transfers with this neor-tagged chromosome into recipient hamster, primate, and canine fibroblasts produced a panel of primary neor transferents, each containing a portion of, or all of, the murine MHC. A cosmid library was made from one such transferent, CHMD(D)B1. Cosmid clones were divided, using species-specific repeat probes, into those containing murine (donor) DNA sequences and those containing sequences derived from the recipient cell. The murine-specific cosmids were clustered into overlapping DNA segments by restriction enzyme digest analysis of the cosmid DNAs coupled with Southern blot analysis with, as probes, murine-specific repeat sequences and nick-translated murine genomic DNA. These cosmid clusters were analyzed for their position within or outside of the MHC, using recombinant mouse strains, and for the presence within them of known murine MHC genes.

Size Fractionation Using Sucrose Gradients

DNA fragments migrate through a linear sucrose gradient at a rate that is dependent on their size. This procedure described in this unit provides good resolution for DNA fragments 5 to 60 kb in size, and sucrose gradients are also useful for purification of bacteriophage l vector arms. Partially digested genomic DNA can be fractionated for the production of cosmid or bacteriophage libraries and completely digested DNA can be fractionated for subgenomic DNA libraries. Protocols are provided in this unit for both partial and complete enzyme digestion of genomic DNA.

Expression of Herpes Simplex Virus Type 1 and Type 2 Glycoprotein D Genes Using the Escherichia Coli lac Promoter

DNA fragments encoding herpes simplex virus type 1 and type 2 glycoprotein D (gD-1 and gD-2, respectively) have been inserted into plasmid vectors and expressed under the transcriptional control of the Escherichia coli lac promoter-operator. The proteins expressed in this system comprised gD sequences fused to a small bacteriophage λ Cro leader (i.e. Cro-gD). Such Cro-gD fusion proteins were found to be intrinsically unstable in E. coli and accumulated to low levels only. We found that fusion of this Cro-gD coding sequence to the 5′ end of a sequence encoding β-galactosidase (β-gal), resulted in high levels of synthesis of Cro-gD-β-gal fusion proteins provided that certain carboxy-terminal gD coding sequences were deleted. These Cro-gD-β-gal proteins accumulated to levels comprising approximately 10% of the total cell protein and were found to form intracellular insoluble aggregates. Insertion of an in-phase amber nonsense codon between the gD and β-gal coding sequences, resulted in synthesis of both Cro-gD and Cro-gD-β-gal proteins in cells containing suppressors. Under these conditions, the Cro-gD amber fragment was stabilized by the Cro-gD-β-gal protein. Chimaeric gD-1 and gD-2 containing proteins were found to be immunologically active and induced antibodies in rabbits which immunoprecipitated authentic gD polypeptides and neutralized the infectivity of both virus types.

Plating and Transferring Cosmid and Plasmid Libraries

There are two commonly used protocols for the screening of recombinant bacteria with hybridization probes. One method involves the spreading of bacteria on the surface of agar using a sterile spreader. A nitrocellulose membrane filter is then placed on top of the colonies and most of each colony is transferred to the filter. This method works well when relatively small numbers of positive colonies are being selected (up to several thousand). A second method, described in this unit, employs a matrix of some type (here nitrocellulose filters are used) upon which bacteria can be plated and grown when the filter is placed on top of a nutrient agar surface. Once the plated bacteria have grown into visible colonies, the filters can be used for replica plating and in situ hybridization analysis.

Amplification of Cosmid and Plasmid Libraries

The purpose of amplification is to provide a reagent library that can be used many times. In this protocol, bacteria containing the recombinant clones are grown on agar plates, washed off the plates, and stored in glycerol. An amplified cosmid or plasmid library will contain one librarys equivalence in 10 to 100 microl. By producing 100 microl of such a library frozen in 100 1-ml tubes, over 1000 platings can be expected.

Purification of Cosmid and Plasmid Clones

Cosmid‐ or plasmid‐bearing colonies that are identified by hybridization are purified by spreading the cosmids or plasmids on an agar plate and repeating the colony hybridization.