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R A Sturm

Publications and source records attributed to R A Sturm.

49 records · Page 3Linked to original sources
PubMed1993

Broad binding-site specificity and affinity properties of octamer 1 and brain octamer-binding proteins.

The ubiquitous Pit-1-Oct-1-Unc-1 (POU)-domain protein octamer 1 (Oct-1) has been observed to bind specifically to a number of degenerate and dissimilar sequences. We have used antibodies directed against a C-terminal Oct-1 peptide to immunoselect binding sequences for HeLa cell Oct-1 from random-sequence oligonucleotides and we describe the isolation of binding sequences of considerable heterogeneity. Although our consensus alignment indicated a 9-bp TATGCAAAT motif with AT-rich flanking sequences, this binding motif is not immediately obvious in the population of sequences and no clone actually contained this sequence. Screening these Oct-1-binding sequences with a mouse whole-brain extract demonstrated that the neuronal octamer-binding proteins exhibit similar but distinct DNA sequence specificities. Unlike the reported selection of binding sequences for other transcription factors, the dependence of Oct-1-binding affinity upon sequence did not correspond tightly to the degree of conservation at particular positions of the consensus sequence. Our results suggest that either base-specific hydrogen bonding is not the only major determinant of binding affinity and specificity, or that Oct-1 binding to some sequences is mechanistically different from its binding to an octamer. These results exemplify the potential to overlook binding sites for some factors by searching gene sequences with a consensus nucleotide sequence.

Animals↗
PubMed1993

Chromosomal structure and expression of the human OTF1 locus encoding the Oct-1 protein.

The genomic structure of the POU domain containing oct-1 gene (OTF1 locus) coding region has been determined using human DNA recombinant bacteriophage and a yeast artificial chromosome clone. The gene is encoded by 16 exons spanning over 150 kb, and the Oct-1 protein reading frame has been extended to 766 amino acids. The exonic structure has been compared to the mouse Oct-2 protein and reveals a conservation of exon-intron boundaries as well as protein sequence similarity. To provide insight into Oct-1 control of transcriptional regulation during the cell-cycle the expression of the oct-1 gene was examined during cellular DNA replication and shows that the steady-state level of the oct-1 mRNA is not S-phase regulated.

Amino Acid Sequence↗
PubMed1993

In vivo and in vitro expression of octamer binding proteins in human melanoma metastases, brain tissue, and fibroblasts.

The pattern of octamer sequence-specific DNA binding proteins expressed in human melanoma was examined in nuclear extracts of seven surgically-isolated tumors, short-term cultures of these tumors, and 25 human melanoma cell lines to determine the in vivo and in vitro distribution of the melanocytic-associated Oct-M1 and Oct-M2 octamer binding activities. In the biopsy tissue and cultured melanoma cells of a metastasis from the cerebellum, two other binding activities (N-Oct-2 and N-Oct-6) in addition to the Oct-M1, Oct-M2 and the generally expressed Oct-1 protein were detected; this profile was consistent with that seen in normal human and mouse brain tissue. Melanoma tissue removed from lymph nodes and cell lines established from them also showed Oct-1, Oct-M1, Oct-M2, and N-Oct-2. N-Oct-2 was distinguished from the comigrating Oct-2A activity by failure to react with Oct-2A-specific antibody. All but one of the 25 melanoma cell lines exhibited Oct-1, Oct-M1, and Oct-M2 and/or N-Oct-2 activity, whereas cultured normal melanocytes expressed only Oct-1 and Oct-M1. In contrast to murine fibroblasts, which express only Oct-1, human fibroblast strains also expressed Oct-2A binding activity, which was confirmed by reactivity with Oct-2A antibody and the presence of Oct-2A mRNA and indicated that Oct-2A has a more general role than that of a lymphoid-specific transcription factor. Overall, the results indicate that expression of neural-specific Oct factors in human melanoma is (1) aberrant compared with normal melanocytes, (2) can be modulated by the surrounding tissue in a brain metastasis, and (3) may be part of the altered program of differentiation accompanying transformation.

Animals↗
PubMed1991

Parallel origins of duplications and the formation of pseudogenes in mitochondrial DNA from parthenogenetic lizards (Heteronotia binoei; Gekkonidae).

Analysis of mitochondrial DNAs (mtDNAs) from parthenogenetic lizards of the Heteronotia binoei complex with restriction enzymes revealed an approximately 5-kb addition present in all 77 individuals. Cleavage site mapping suggested the presence of a direct tandem duplication spanning the 16S and 12S rRNA genes, the control region and most, if not all, of the gene for the subunit 1 of NADH dehydrogenase (ND1). The location of the duplication was confirmed by Southern hybridization. A restriction enzyme survey provided evidence for modifications to each copy of the duplicated sequence, including four large deletions. Each gene affected by a deletion was complemented by an intact version in the other copy of the sequence, although for one gene the functional copy was heteroplasmic for another deletion. Sequencing of a fragment from one copy of the duplication which encompassed the tRNA(leu)(UUR) and parts of the 16S rRNA and ND1 genes, revealed mutations expected to disrupt function. Thus, evolution subsequent to the duplication event has resulted in mitochondrial pseudogenes. The presence of duplications in all of these parthenogens, but not among representatives of their maternal sexual ancestors, suggests that the duplications arose in the parthenogenetic form. This provides the second instance in H. binoei of mtDNA duplication associated with the transition from sexual to parthenogenetic reproduction. The increased incidence of duplications in parthenogenetic lizards may be caused by errors in mtDNA replication due to either polyploidy or hybridity of their nuclear genomes.

Amino Acid Sequence↗
PubMed1991

A melanoma octamer binding protein is responsive to differentiating agents.

Analysis of human melanocytes and melanoma cell lines for proteins interacting with the octamer control sequence (ATGCAAAT) has revealed two distinct melanoma octamer binding proteins, Oct-M1 and Oct-M2. The latter was restricted to cell lines derived from tumor metastases. The level of Oct-M1 activity in a pigmented melanoma line was enhanced in comparison to the general octamer binding protein Oct-1 when cells were cultured in the presence of the depigmenting agent dithiothreitol and conversely was reduced by the differentiating and pigment inducing agents butyric acid and dimethyl sulfoxide.

Butyrates↗
PubMed1988

The POU domain is a bipartite DNA-binding structure.

The POU domain (pronounced 'pow') is a highly charged 155-162-amino-acid (aa) region of sequence similarity contained within three mammalian transcription factors. Pt-1 (ref. 2), Oct-1 (ref. 3) and Oct-2 (ref. 4), and the product of the nematode gene unc-86 (ref. 5) which is involved in determining neural cell lineage. This domain consists of two subdomains, a C-terminal homoeo domain and an N-terminal POU-specific region separated by a short nonconserved linker; the sequence relationship shows that the POU homoeo domains form a distinct POU-related family. In the ubiquitous and lymphoid-specific octamer-motif binding proteins Oct-1 and Oct-2, the POU domain is sufficient for sequence-specific DNA binding. Homoeobox domains contain a helix-turn-helix DNA-binding motif, first identified in bacterial repressors. The helix-turn-helix region of the POU domain is important for DNA binding and, in other classes of homoeo-containing proteins, the entire homoeo domain is sufficient for DNA binding; thus the new POU-specific region could be involved in other functions such as protein-protein interactions. Nevertheless, we show here that in fact the POU domain is a novel bipartite DNA-binding structure in which the POU homoeo and POU-specific regions form two subdomains that are both required for DNA binding but are held together by a flexible linker.

Base Sequence↗
PubMed1988

Conservation of histone H2A/H2B intergene regions: a role for the H2B specific element in divergent transcription.

The organization and function of potential regulatory elements associated with the promoters of chicken H2A and H2B genes pairs have been examined. The intergene regions of six dispersed and divergently-transcribed H2A/H2B gene pairs contain several extremely well conserved and spaced blocks of sequence homology. Adjacent coding regions are on average 342 base-pairs apart. Respective TATA boxes are separated by 180 base-pairs and within this confined region there are four CCAAT boxes and a previously identified 13 base-pair H2B-specific element (H2B-box) which has homology to the octamer motif present in a number of gene promoter/enhancer elements. Transcription of H2A and H2B genes from wild-type and mutant constructs was measured in transient assays by transfection into HeLa cells, and in permanently transformed clonal cell lines. In vitro separation of the two genes at a unique intergenic site significantly decreased transcription of each gene. This suggested that the H2A/H2B gene pairs contained overlapping promoters. Deletion or point mutagenesis of the H2B-specific element decreased the levels of H2B and the H2A transcripts indicating that this sequence is a common regulatory element of both genes in the divergent-pair configeration.

Animals↗
PubMed1988

The ubiquitous octamer-binding protein Oct-1 contains a POU domain with a homeo box subdomain.

The octamer motif ATGCAAAT is recognized indistinguishably by two mammalian transcription factors: one that is expressed ubiquitously and referred to here as Oct-1, and another, Oct-2, that is expressed in lymphoid cells. We report the cDNA cloning of the human oct-1 gene, which encodes Oct-1, by screening lambda gt11 recombinant phage in situ for octamer motif-specific DNA binding. One lambda gt11 recombinant expressed a beta-galactosidase-octamer-binding fusion protein with a DNA-binding specificity indistinguishable from human HeLa cell Oct-1 protein. As expected for a ubiquitously expressed protein, Oct-1 mRNA is expressed in all five human and two mouse cell lines tested. Polyclonal rabbit antiserum raised against the beta-galactosidase fusion protein shows that the DNA-binding domains of Oct-1 and Oct-2 proteins are related antigenically. Deletion analysis of the 743-amino-acid-long oct-1 open reading frame shows that the DNA-binding activity lies within a central highly charged domain of 160 amino acids. Comparison of the Oct-1 and Oct-2 sequences reveals that this domain is nearly identical between the two proteins. Highly similar domains are also present in the pituitary-specific transcription factor Pit-1 and the Caenorhabditis elegans unc-86 cell lineage gene product (see Herr et al. 1988). Within this shared POU (Pit-1, Oct-1 and Oct-2, unc-86) domain (pronounced 'pow') lie two subdomains: a POU-related homeo box and a POU-specific box. The Oct-1 protein is unique among the POU-related proteins and other homeo box proteins because it is expressed ubiquitously.

Amino Acid Sequence↗