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L Dailey

Publications and source records attributed to L Dailey.

31 records · Page 2Linked to original sources
PubMed1988

Purification of the human histone H4 gene-specific transcription factors H4TF-1 and H4TF-2.

Transcription of the cell-cycle-regulated human histone genes increases approximately fivefold during S phase. One step toward the elucidation of the biochemical mechanisms that govern cell-cycle-regulated expression of these genes is to purify and characterize the transcription factors that regulate these promoters. Here, we describe the purification of two previously identified factors, H4TF-1 and H4TF-2, which bind the human histone H4 promoter. Purification was achieved through a combination of ion-exchange and oligonucleotide affinity chromatography. On the basis of analysis of purified fractions by SDS-polyacrylamide gels and UV cross-linking, we believe that H4TF-1 is two polypeptides of 105 and 110 kD. This factor binds to a GC-rich DNA sequence required for maximal expression of the H4 gene but does not bind to any Sp1 consensus elements tested. H4TF-2 is a 65-kD protein that binds specifically to sequences within that highly conserved H4 subtype-specific consensus promoter element. Both highly purified factors activated transcription in vitro only from H4 promoters that contained their binding sequences, demonstrating that H4TF-1 and H4TF-2 are H4-specific transcription factors that potentiate expression of this gene.

Base Sequence↗
PubMed1987

RNA polymerase II transcription factors H4TF-1 and H4TF-2 require metal to bind specific DNA sequences.

Specific DNA-binding and in vitro transcription activities of H4TF-1 and H4TF-2 are inactivated by chelating agents. Binding activity is restored by addition of Zn2+, and H4TF-2 is also reactivated by Fe2+. In contrast, preformed factor-DNA complexes are resistant to chelators. Therefore, metal ions are a required component of the H4TF-1 and H4TF-2 DNA-binding domains.

Cations, Divalent↗
PubMed1986

Distinct transcription factors bind specifically to two regions of the human histone H4 promoter.

Two proteins specifically binding to separate regions of the human histone H4 promoter were identified in nuclear extracts prepared from synchronized S-phase HeLa cells. Competition experiments with H4 promoter mutants and DNase protection assays ("footprinting") demonstrate that these factors bind to regions of the H4 promoter that are essential for maximal expression in vitro. One of these factors (H4TF-1) binds to sequences between -80 and -110 base pairs upstream of the H4 cap site, whereas the other (H4TF-2) binds to the H4 subtype-specific sequence element immediately upstream from the "TATA" homology. Neither of these activities can efficiently bind to any of the other histone gene subtypes or simian virus 40 DNA. Binding of H4TF-1 to the distal region of the pHu4A histone H4 promoter is inhibited competitively with varying efficiency by four of six human histone H4 genes cloned in this laboratory, whereas efficient competition for binding of H4TF-2 is exhibited by five of the six H4 genes. Since both of these factors bind to significant regions of the pHu4A histone H4 promoter and can be bound by several different human H4 genes, we believe that they are important for maximal transcription of the gene and that they may be involved in its regulated expression during the cell cycle.

Binding, Competitive↗
PubMed1985

Sequences in the polyomavirus DNA regulatory region involved in viral DNA replication and early gene expression.

We constructed and analyzed a series of deletion mutants in the noncoding regulatory region of tsa polyomavirus DNA to identify some of the sequences critical to the DNA replication origin and to the expression of the viral early genes in vivo. By using both transient and long-term assays under conditions where the influence of large T antigen (T-Ag) in replication or autoregulation was minimized, we observed no more than a 30% reduction in early gene expression upon removal of the CAAT or TATA elements or both. These assays demonstrated a predominant effect of upstream promoter or enhancer elements and indicated that removal of the CAAT or TATA boxes did not significantly affect viral early gene expression. Studies on the replicative ability of these mutants in mouse cells constitutively expressing the polyoma early proteins revealed that the removal of DNA sequences contained within a previously identified T-Ag high-affinity binding site (nucleotides 39 to 64) abolished viral DNA replication, whereas removal of two other high-affinity sites, closer to the early mRNA cap sites, did not. Furthermore, a deletion including this same high-affinity site plus a low-affinity binding site within the 32-base-pair palindrome of the origin core sequences eliminated the ability of the viral large T-Ag to efficiently repress early gene transcription. It is thus possible that the origin-proximal high-affinity T-Ag binding site is involved in both of the functions of large T-Ag, i.e., the initiation of viral DNA replication and the autoregulation of early gene transcription.

Animals↗
PubMed1985

Common regulatory elements control gene expression from polyoma early and late promoters in cells transformed by chimeric plasmids.

In a previous report we showed that transcripts initiating from the late promoter of integrated polyoma plasmids could be detected at significant levels when neomycin resistance (neo) coding sequences were linked to this promoter. In this report we used chimeric plasmids that contain either a limited portion of the polyoma genome or deletions within the polyoma noncoding regulatory region to determine the sequence requirements for late promoter activity in this system. We observed no absolute requirement for either the polyoma early coding region or the origin of DNA replication for Neo-r colony formation. We were therefore able to independently assess the effects of deletions in the polyoma enhancer region on gene activity in both the early and late directions. We measured the ability of cells transfected with plasmids containing deletions in this region to form colonies in either semisolid or G418-containing medium under nonreplicative conditions. Our results indicate that either the PvuII 4 fragment, which contains the simian virus 40 core enhancer sequence, or a region from nucleotides 5099 to 5142, which contains the adenovirus type 5 E1A core enhancer sequence, can be deleted without significantly affecting gene expression in either direction. However, a deletion of nucleotides 5099 to 5172 reduced activities to similar extents in both directions, and a plasmid containing a larger deletion of nucleotides 5055 to 5182 showed a further reduction in activity. Although having no effect by itself, a second origin region deletion of nucleotides 5246 to 127 when present in these mutant backgrounds caused either a further reduction or elimination, respectively, of both G418 and agar colony-forming ability, suggesting the presence of an additional common regulatory element within this region. A comparison of 5' ends of neo transcripts present in cells transformed by these plasmids suggested that the reduction in activity was due to deletion of regulatory rather than structural elements of the late promoter. Our results indicate that the noncoding region of polyoma contains multiple complementing regulatory elements that control the level of both early and late gene expression.

Animals↗
PubMed1984

Amplification and excision of integrated polyoma DNA sequences require a functional origin of replication.

Cells transformed by Polyoma virus (Py) can undergo a high rate of excision or amplification of integrated viral DNA sequences, and these phenomena require the presence of homology (i.e., repeats) within the viral insertion as well as a functional viral large T antigen (T-Ag). To determine whether the main role of large T-Ag in excision and amplification was replicative or recombination-promoting, we studied transformed rat cell lines containing tandem insertions of a ts-a Py molecule (encoding a thermolabile large T-Ag) with a deletion of the origin of viral DNA replication. Culturing of these cells at the temperature permissive for large T-Ag function did not result in any detectable excision or amplification of integrated Py sequences. We then introduced into origin-defective lines a recombinant plasmid containing the viral origin of replication and the gene coding for resistance to the antibiotic G418. All G418-resistant clones analyzed readily amplified the integrated plasmid molecules when grown under conditions permissive for large T-Ag function, showing that these cells produced viral large T-Ag capable of promoting amplification in trans of DNA sequences containing the Py origin. These observations strongly suggest that Polyoma large T antigen promotes excision or amplification of viral DNA by initiating replication at the integrated origin, providing a favorable substrate for subsequent recombination.

Animals↗
PubMed1984

Deletion of the origin of replication impairs the ability of polyomavirus DNA to transform cells and to form tandem insertions.

We examined the transforming properties of polyomavirus DNA molecules which can produce a functional large T-antigen but which are cis defective for viral DNA replication. The inability of these molecules to replicate results from the deletion of sequences comprising the viral replication origin. We found that even in the presence of a functional large T-antigen, transformation of rat cells by these viral DNAs was greatly reduced when compared with replication-competent parental DNA, and cells transformed by origin-minus mutants generally contained the integrated viral DNA in a nontandem arrangement. Therefore, polyomavirus large T-antigen promotes the establishment of transformation and tandem integration by interacting with the viral origin of DNA replication. This indicates that viral DNA synthesis is directly involved in these processes.

Animals↗
PubMed1982

Requirements for excision and amplification of integrated viral DNA molecules in polyoma virus-transformed cells.

The integration of polyoma virus DNA into the genome of transformed rat cells generally takes place in a tandem head-to-tail arrangement. A functional viral large tumor antigen (T-Ag) renders this structure unstable, as manifested by free DNA production and excision or amplification of the integrated viral DNA. All of these phenomena involve the mobilization of precise genomic "units," suggesting that they result from intramolecular homologous recombination events occurring in the repeated viral DNA sequences within the integrated structures. We studied polyoma ts-a-transformed rat cell lines, which produced large T-Ag but contained less than a single copy of integrated viral DNA. In all of these lines, reversion to a normal phenotype (indicative of excision) was extremely low and independent of the presence of a functional large T-Ag. The revertants were either phenotypic or had undergone variable rearrangements of the integrated sequences that seemed to involve flanking host DNA. In two of these cell lines (ts-a 4A and ts-a 3B), we could not detect any evidence of amplification even after 2 months of propagation under conditions permissive for large T-Ag. An amplification event was detected in a small subpopulation of the ts-a R5-1 line after 2 months of growth at 33 degrees C. This involved a DNA fragment of 5.1 kilobases, consisting of the left portion of the viral insertion and about 2.5 kilobases of adjacent host DNA sequences. None of these lines spontaneously produced free viral DNA, but after fusion with 3T3 mouse fibroblasts, R5-1 and 4A produced a low level of heterogeneous free DNA molecules, which contained both viral and flanking host DNA. In contrast, the ts-a 9 cell line, whose viral insertion consists of a partial tandem of approximately 1.2 viral genomes, underwent a high rate of excision or amplification when propagated at temperatures permissive for large T-Ag function. These results indicate that the high rate of excision and amplification of integrated viral genomes observed in polyoma-transformed rat cells requires the presence of regions of homology (i.e., repeats) in the integrated viral sequences. Therefore, these events occur via homologous intramolecular recombination, which is promoted directly or indirectly by the large viral T-Ag.

Animals↗
PubMed1980

Amplification of integrated viral DNA sequences in polyoma virus-transformed cells.

Polyoma virus (Py) transformation of rat cells requires integration of viral genomes into the host DNA, which generally occurs in a partial or full head-to-tail tandem arrangement. The instability of this structure was previously demonstrated by the high rate of loss of integrated Py genomes in the presence of viral large tumor (T) antigen. We now show that integrated Py DNA sequences can also undergo amplification. We studied two rat cell lines transformed by the ts-a Py mutant, which codes for a thermolabile large T antigen. In a derivative of the ts-a H6A cell line, we have observed loss of full-length Py DNA molecules from the integrated tandem ("curing"), accompanied by the creation of new tandem repeats of two segments of viral DNA corresponding to 38% and 10% of the viral genome, each containing the origin of DNA replication. In the ts-a H3A cell line, which contains an integrated partial tandem of about 1.3 viral genomes with three distinct deletions, propagation at 33 degrees C resulted in the generation of full tandem repeats of a 94% Py DNA "unit" (including two 3% deletions), an 85% "unit" (including a 3% and the 12% deletion), or both. Amplification of integrated viral DNA was not observed in cells propagated at 39.5 degrees C, the nonpermissive temperature for large T antigen function. Amplification of integrated Py DNA sequences thus requires an active large T antigen and can generate a full tandem of integrated viral DNA molecules long after the initial integration event.

Antigens, Neoplasm↗
PubMedDate not supplied

Latissimus dorsi transfer to restore elbow extension in obstetrical palsy.

Four patients with weak triceps muscle function, secondary to obstetrical palsy, were treated by transfer of the latissimus dorsi on its neurovascular pedicle to provide effective elbow extension. The average age of the patients at surgery was 10 years, and follow-up ranged from 12 to 53 months. Postoperatively, all patients demonstrated increased strength of elbow extension and improvement in activities of daily living. These satisfactory results support the use of latissimus dorsi transfer in patients with weak elbow extension secondary to obstetrical palsy.

Adolescent↗
PubMedDate not supplied

Use of the Green transfer in treatment of patients with spastic cerebral palsy: 17-year experience.

We retrospectively reviewed Green procedures and transfer of the flexor carpi ulnaris (FCU) in treatment of spastic forearm pronation, wrist volarflexion, and ulnar deviation deformities. Patient's ages ranged from 3 years 5 months to 16 years 5 months. Surgically, a single volar incision was made and the extensor carpi radialis brevis and/or longus (ECRB, ECRL) were used for insertion. The FCU was tensioned at neutral against gravity and immobilized in 5 degrees of dorsiflexion and 45 degrees of supination. Arc of flexion (47 degrees) did not change but was centered about neutral. Supination improved markedly when operation included a pronator procedure. Quadraplegia athetosis and intellectual impairment did not affect the operative result adversely. Patients aged greater than 12 years showed less functional improvement. Eighty-eight percent had cosmetic improvement, and 79% improved functionally. None had a decreased functional rating.

Adolescent↗