Faculty Bibliography

The lamin B receptor (LBR) is a polytopic integral membrane protein localized exclusively in the inner nuclear membrane domain of the nuclear envelope. Its cDNA deduced primary structure consists of a highly charged amino-terminal domain of 205 residues that faces the nucleoplasm followed by a hydrophobic domain with eight potential transmembrane segments. To identify determinants that sort LBR from its site of integration (RER and outer nuclear membrane) to the inner nuclear membrane, we prepared full-length, truncated, and chimeric cDNA constructs of chick LBR, transfected these into mammalian cells and detected the expressed protein by immunofluorescence microscopy using appropriate antibodies. Surprisingly, we found that the determinants for sorting of LBR to the inner nuclear membrane reside in a region comprising its first transmembrane sequence plus flanking residues on either side. The other transmembrane regions as well as the nucleoplasmic domain are not required for sorting. We propose that the first transmembrane segment of LBR interacts specifically with another transmembrane segment and consider several mechanisms by which such specific interaction could result in sorting to the inner nuclear membrane.

Chromatin assembly factor I (CAF-I) is a multisubunit protein complex purified from the nuclei of human cells and required for chromatin assembly during DNA replication in vitro. Purified CAF-I promotes chromatin assembly in a reaction that is dependent upon, and coupled with, DNA replication and is therefore likely to reflect events that occur during S phase in vivo. In order to investigate the regulation and mechanism of CAF-I and the replication-dependent chromatin assembly process, we have used the purified protein to raise monoclonal antibodies. In this report we describe the characterization of a panel of monoclonal antibodies which recognize different subunits of the CAF-I complex. We use immunoprecipitation analysis to show that CAF-I exists as a multiprotein complex in vivo and that some of the polypeptides are phosphorylated. In addition, immunocytochemistry demonstrates that CAF-I is localized to the nucleus of human cells. Finally, monoclonal antibodies directed against the individual subunits of CAF-I immunodeplete chromatin assembly activity from nuclear extracts, confirming that CAF-I is a multisubunit protein required for chromatin assembly in vitro.

A cell free system that supports replication-dependent chromatin assembly has been used to determine the mechanism of histone deposition during DNA replication. CAF-I, a human cell nuclear factor, promotes chromatin assembly on replicating SV40 DNA in the presence of a crude cytosol replication extract. Biochemical fractionation of the cytosol extract has allowed separation of the chromatin assembly reaction into two steps. During the first step, CAF-I targets the deposition of newly synthesized histones H3 and H4 to the replicating DNA. This reaction is dependent upon and coupled with DNA replication, and utilizes the newly synthesized forms of histones H3 and H4, which unlike bulk histone found in chromatin, do not bind to DNA by themselves. The H3/H4-replicated DNA complex is a stable intermediate which exhibits a micrococcal nuclease resistant structure and can be isolated by sucrose gradient sedimentation. In the second step, this replicated precursor is converted to mature chromatin by the addition of histones H2A and H2B in a reaction that can occur after DNA replication. The requirement for CAF-I in at least the first step of the reaction suggests a level of cellular control for this fundamental process.

The purification and characterization of a replication-dependent chromatin assembly factor (CAF-I) from the nuclei of human cells is described. CAF-I is a multisubunit protein that, when added to a crude cytosol replication extract, promotes chromatin assembly on replicating SV40 DNA. Chromatin assembly by CAF-I requires and is coupled with DNA replication. The minichromosomes assembled de novo by CAF-I consist of correctly spaced nucleosomes containing the four core histones H2A, H2B, H3, and H4, which are supplied in a soluble form by the cytosol replication extract. Thus, by several criteria, the CAF-I-dependent chromatin assembly reaction described herein reflects the process of chromatin formation during DNA replication in vivo.

Cloned DNA fragments encoding portions of canine parvovirus (CPV) structural proteins were inserted into plasmid expression vectors. These plasmids expressed CPV-beta-galactosidase fusion proteins under the transcriptional control of the Escherichia coli lac promoter-operator. The fusion proteins were purified and used to immunize rabbits. Rabbit antibodies raised against these fusion proteins were shown to immunoprecipitate authentic CPV structural proteins from infected cell extracts. This demonstrated that the CPV-beta-galactosidase fusion proteins expressed in bacteria elicit antibodies which can recognize determinants of authentic CPV proteins. However, none of the antibodies neutralizes CPV virus particles.

The expression of the two nonallelic but highly homologous rat insulin genes (1 and 2) in a transplantable beta-cell tumor is found to be 10-fold higher for rat1 insulin than rat2 insulin, while in normal pancreatic tissue there are approximately equal amounts of each protein. No large sequence rearrangements of the genes were apparent by restriction analysis of the tumor DNA, and both genes were found to be specifically hypomethylated in the tumor as compared with other nonpancreatic tissue. Equivalent amounts of both insulin 1 and 2 precursor transcripts, as well as stable, mature mRNAs were detected in the tumor. However, two-dimensional gel analysis of immunoprecipitated rat1 and rat2 preproinsulins synthesized in vitro revealed a 10:1 ratio of rat1 to rat2 proteins. A 1:1 ratio was obtained when the tumor mRNA was treated in vitro with vaccinia virus capping extract, suggesting a structural modification at the 5' terminus of the rat2 mRNA. These results are discussed in the context of insulin regulation by glucose, shown to be due to translational control.

The present communication describes the molecular cloning and DNA sequence determination of the rat growth hormone (rGH) gene. The rGH gene was cloned on an 11 kilobase EcoRI fragment of total rat DNA; it has four intervening sequences which correspond in position to those of the human growth hormone (hGH) gene. One of the intervening sequences in the rGH gene contains a possible transposable element: a 200 base pair direct repeat that is itself flanked by an exact 15 base pair direct repeat. The DNA sequence was used to estimate the location of the 5' end of the mature growth hormone mRNA. By S1 nuclease mapping it was located approximately 25 bases "downstream" from a TATAAA sequence presumed to play a role in initiation of transcription of the rGH gene.