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Biomedical subjects

C Queen

Publications and source records attributed to C Queen.

49 records · Page 3Linked to original sources
PubMed1982

Expression of an Abelson murine leukemia virus-encoded protein in Escherichia coli causes extensive phosphorylation of tyrosine residues.

A segment of the Abelson murine leukemia virus (A-MuLV) genome was inserted into an Escherichia coli plasmid designed to allow the expression of the protein encoded by the viral gene. Bacteria expressing the A-MuLV-encoded protein were isolated; they had new phosphorylated proteins in which the phosphate was linked to tyrosine residues. These proteins included many that must be E. coli protein. One phosphotyrosine-containing protein of 62,000 molecular weight had reactivity with antiserum specific for authentic A-MuLV protein. The A-MuLV protein thus appears to be a tyrosine-specific protein kinase which is active in E. coli.

Abelson murine leukemia virus↗
PubMed1981

Interspersed repeated sequences in the African green monkey genome that are homologous to the human Alu family.

The dominant family of interspersed repetitive DNA sequences in the human genome has been termed the Alu family. We have found that more than 75% of the lambda phage in a recombinant library representing an African green monkey genome hybridize with a human Alu sequence under stringent conditions. A group of clones selected from the monkey library with probes other than the Alu sequence were analyzed for the presence and distribution of Alu family sequences. The analyses confirm the abundance of Alu sequences and demonstrate that more than one repeat unit is present in some phages. In the clones studied, the Alu units are separated by an average of 8 kilobase pairs of unrelated sequences. The nucleotide sequence of one monkey Alu sequence is reported and shown to resemble the human Alu sequences closely. Hence, the sequence, dispersion pattern, and copy number of the Alu family members are very similar in the African green monkey and human genomes. Among the clones investigated were two that contain segments of the satellite DNA term alpha-component joined to non alpha-component DNA. The experiments indicate that in the monkey genome Alu sequences can occur close to regions of alpha-component DNA.

Animals↗
PubMed1981

A promoter of pBR322 activated by cAMP receptor protein.

We have demonstrated in vitro the existence on the plasmid pBR322 of a promoter signal that is strictly dependent on cAMP and its receptor protein CRP. Transcription initiates with pppG at nucleotide 2270 and proceeds counterclockwise on the standard pBR322 map. DNase protection studies show that CRP selectively binds to the -35 region of the promoter. This region exhibits strong structural homologies to the binding sites of other CRP-dependent promoters.

Base Sequence↗
PubMed1981

Differential translation efficiency explains discoordinate expression of the galactose operon.

We have used an mRNA-dependent E. coli S-30 translation system to compare the translation efficiencies of two polycistronic transcripts of the galactose operon, the CRP-cAMP-dependent mRNA (P1) and the CRP-cAMP-independent mRNA (P2). The RNAs were prepared in vitro, quantitated by hybridization or gel analysis and translated in a cell free system. The specific protein products were measured, and their quantities were compared with the amount of input mRNA. Our results show that the P2 mRNA synthesizes epimerase, the 5'-proximal gene product of the gal operon, four times more efficiently than the P1 mRNA. The 5'-distal gene products, transferase and kinase, are translated with the same efficiency from both transcripts. Thus the ratio of epimerase to kinase synthesis is four times higher for the P2 mRNA than for the P1 mRNA. This change in epimerase to kinase ratio is identical to that observed in vivo when the cellular cAMP level falls and gal transcription is believed to switch from P1 to P2. We suggest that it is the differential translation efficiency of the epimerase gene on the two different gal transcripts that accounts for this discoordinate expression. Moreover, since the P2 mRNA differs from the P1 mRNA only by the addition of five nucleotides at the 5' terminus and these nucleotides are outside the ribosome binding region we determine for epimerase, the selective difference in the translation efficiency of epimerase is probably mediated by RNA conformation.

Carbohydrate Epimerases↗
PubMed1981

Three segments from the monkey genome that hybridize to simian virus 40 have common structural elements.

Three cloned segments that hybridize to a region of simian virus 40 (SV40) deoxyribonucleic acid including the origin of replication have been isolated from a monkey genomic library. The primary structure of one segment was previously reported (T. McCutchan and M. Singer, Proc. Natl. Acad. Sci. U.S.A. 78:95-99, 1981). We report here the sequences of the other two segments and a comparison of all three. The SV 40-hybridizing region in each segment is limited to several hundred base pairs. All of the segments contain multiple and disconnected sequences homologous to the region of SV40 directly surrounding the viral replication origin. The number and arrangement of the homologous sequences is different in the three segments. However, the segments have the following features in common: (i) each contains multiple copies of the sequence GGGCGGPuPu, which also appears six times near the origin of SV40; (ii) each contains several strong homologies to the central dyad symmetry of SV40; (iii) each contains a long internal repeat, as does the origin region of SV40. The three SV40-hybridizing segments are members of a larger family of genomic sequences that hybridize well to each other, but not necessarily to SV40.

Animals↗
PubMedDate not supplied

Comparison of the three-dimensional structures of a humanized and a chimeric Fab of an anti-gamma-interferon antibody.

The objective of this work is to compare the three-dimensional structures of "humanized" and mouse-human chimeric forms of a murine monoclonal antibody elicited against human gamma-interferon. It is also to provide structural explanations for the small differences in the affinities and biological interactions of the two molecules for this antigen. Antigen-binding fragments (Fabs) were produced by papain hydrolysis of the antibodies and crystallized with polyethylene glycol (PEG) 8,000 by nearly identical microseeding procedures. Their structures were solved by X-ray analyses at 2.9 A resolution, using molecular replacement methods and crystallographic refinement. Comparison of these structures revealed marked similarities in the light (L) chains and near identities of the constant (C) domains of the heavy (H) chains. However, the variable (V) domains of the heavy chains exhibited substantial differences in the conformations of all three complementarity-determining regions (CDRs), and in their first framework segments (FR1). In FR1 of the humanized VH, the substitution of serine for proline in position 7 allowed the N-terminal segment (designated strand 4-1) to be closely juxtaposed to an adjacent strand (4-2) and form hydrogen bonds typical of an antiparallel beta-pleated sheet. The tightening of the humanized structure was relayed in such a way as to decrease the space available for the last portion of HFR1 and the first part of HCDR1. This compression led to the formation of an alpha-helix involving residues 25-32. With fewer steric constraints, the corresponding segment in the chimeric Fab lengthened by at least 1 A to a random coil which terminated in a single turn of 310 helix. In the humanized Fab, HCDR1, which is sandwiched between HCDR2 and HCDR3, significantly influenced the structures of both regions. HCDR2 was forced into a bent and twisted orientation different from that in the chimeric Fab, both at the crown of the loop (around proline H52a) and at its base. As in HCDR1, the last few residues of HCDR2 in the humanized Fab were compressed into a space-saving alpha-helix, contrasting with a more extended 310 helix in the chimeric form. HCDR3 in the humanized Fab was also adjusted in shape and topography. The observed similarities in the functional binding activities of the two molecules can be rationalized by limited induced fit adjustments in their structures on antigen binding. While not perfect replicas, the two structures are testimonials to the progress in making high affinity monoclonal antibodies safe for human use.

Amino Acid Sequence↗
PubMedDate not supplied

Cell-type specific expression of a transfected immunoglobulin gene.

The introduction of cloned genes into eukaryotic cells has become a major technique in the study of gene expression. Many experiments have demonstrated transcription of cloned genes after transfection into heterologous cell systems--cells in which the genes are not normally active. More recently, several investigators have obtained expression of specialized genes after transfection into cells of the corresponding specialized type, notably the beta-globin gene in erythroleukaemic cells and immunoglobulin genes in myeloma cells. These results allow the study of gene expression during development by comparing transcription of a gene transfected into homologous and heterologous cells. We have shown that a rearranged kappa immunoglobulin gene, cloned from a mouse myeloma, is transcribed transiently at a high level when reintroduced into mouse myeloma cells. We show here, in an internally controlled manner, that the same immunoglobulin gene is not detectably transcribed when transfected into mouse 3T3 or L cells.

Animals↗
PubMedDate not supplied

An immunoglobulin promoter displays cell-type specificity independently of the enhancer.

Recent studies in which cloned immunoglobulin genes were introduced into cultured cells have produced two significant findings. First, the genes are expressed after transfection into lymphoid cells but not non-lymphoid cells. Second, transcription of an immunoglobulin gene requires, in addition to the promoter region, an enhancer element located downstream of the transcription start site. These findings raise the question of whether it is the promoter or the enhancer region that is responsible for the observed cell-type specificity. It has, in fact, been shown that immunoglobulin enhancers function only in lymphoid cells. We show here that the promoter for an immunoglobulin kappa light-chain gene also is strongly specific for lymphoid cells. Our result reemphasizes the importance of promoters relative to enhancers in determining which cells express which genes.

Animals↗
PubMedDate not supplied

Synergism between immunoglobulin enhancers and promoters.

Enhancers are DNA sequences that stimulate transcription from eukaryotic promoters. This stimulatory effect can be exerted over large distances and from a position either 5' or 3' of a promoter. Enhancers have been found in the genomes of many viruses, and in some cellular genes such as those encoding immunoglobulin heavy chain and kappa light chain. An important feature of both viral and cellular enhancers is the ability of each enhancer to stimulate transcription from many promoters other than the one with which it is found associated. However, the question of whether cellular enhancers stimulate their 'own' promoter more efficiently than other promoters has apparently not been investigated. We show here that the kappa light-chain enhancer stimulates a kappa promoter about 20-fold more than it stimulates either the simian virus 40 (SV40) early promoter or a metallothionein (MT) promoter, two promoters that are very sensitive to other enhancers. Similarly, the heavy-chain enhancer stimulates a heavy-chain promoter much more than it stimulates the SV40 and MT promoters. This synergism between immunoglobulin enhancers and promoters might be due to the action of a protein that binds specifically to each of the regulatory elements.

Animals↗