Paleocoastal marine fishing on the Pacific Coast of the Americas: perspectives from Daisy Cave, California.

Analysis of over 27,000 fish bones from strata at Daisy Cave dated between about 11,500 and 8500 cal B.P. suggests that early Channel Islanders fished relatively intensively in a variety of habitats using a number of distinct technologies, including boats and the earliest evidence for hook-and-line fishing on the Pacific Coast of the Americas. The abundance of fish remains and fishing-related artifacts supports dietary reconstructions that suggest fish provided more than 50 percent of the edible meat represented in faunal samples from the early Holocene site strata. The abundance and economic importance of fish at Daisy Cave, unprecedented among early sites along the Pacific Coast of North America, suggest that early maritime capabilities on the Channel Islands were both more advanced and more variable than previously believed. When combined with a survey of fish remains from several other early Pacific Coast sites, these data suggest that early New World peoples effectively used watercraft, captured a diverse array of fish, and exploited a variety of marine habitats and resources.

Oligonucleotide-capped gold nanoparticles for improved atomic force microscopic imaging and enhanced selectivity in polynucleotide detection.

A novel assay for selective determination of polynucleotides using atomic force microscopy in conjunction with the formation of the probe/target/DNA-gold nanoparticle sandwich structure at a gold surface is described. A 17-mer probe was attached to the surface for subsequent hybridization with a polynucleotide target. Due to the flat orientation of the probe-target hybrid with respect to the surface and the spatial obstruction of the unhybridized probes near the hybrids, the AFM images are not clear. The hybridization efficiency was estimated to be about 1.1% since certain surface features could not be resolved. The utilization of 30-mer-capped gold nanoparticles not only provides another dimension of selectivity, but also reorients the previously formed probe-target hybrid in such a way that the strands of the target become tethered with respect to the surface. This reorientation improves the resolution in imaging the hybridized target molecules and provides an accurate determination of the hybridization efficiency (16%).

Functional role of a conformationally flexible homopurine/homopyrimidine domain of the androgen receptor gene promoter interacting with Sp1 and a pyrimidine single strand DNA-binding protein.

The androgen receptor (AR) gene promoter does not contain the TATA or CAAT box, but it contains a long (approximately 90-bp) homopurine/homopyrimidine (pur/ pyr) stretch immediately upstream of the Sp1-binding GC box site. This pur-pyr stretch is conserved at the same proximal position in the rat, mouse, and human AR gene promoters. Mutation of this region results in a 3-fold decline in promoter activity, indicating an important regulatory function. Examination of the conformational state of the AR pur/pyr region with the single-strand-specific S1 nuclease showed that it is capable of forming a non-B DNA structure involving unpaired single strands. Fine mapping of the S1-sensitive site revealed an unsymmetric cleavage pattern indicative of an intramolecular triple helical H-form DNA conformation. Electrophoretic mobility shift analyses showed that the pur/pyr region of the AR promoter can bind a novel pyrimidine single-strand-specific protein (ssPyrBF) and also a double-strand DNA-binding protein. Both oligonucleotide cross-competition and antibody supershift experiments established that the double-strand binding protein is equivalent to Sp1. Deoxyribonuclease I (DNase I) footprinting analysis showed multiple Sp1-binding to the pur/pyr site and a weaker Sp1 interaction to this region compared with the adjacently located GC box, where Sp1 functions to recruit the TFIID complex. These results suggest that the pur/pyr domain of the AR gene can serve to attract additional Sp1 molecules when it exists in the double-stranded B-DNA conformation. However, binding of ssPyrBF and the resultant stabilization of the non-B DNA structure is expected to prevent its interaction with Sp1. We speculate that in the TATA-less AR gene promoter, multiple weak Sp1 sites at the pur/pyr region adjacent to the GC box can provide a readily available source of this transcription factor to the functional GC box, thereby facilitating the assembly of the initiation complex.

Age-dependent expression of the androgen receptor gene in the prostate and its implication in glandular differentiation and hyperplasia.

The senescence phenotype is the product of both cumulative physical damages during the life span and a species-specific genetic program. The genetic program of aging appears to have co-evolved with the sexual mode of reproduction. The same developmental processes that prepare the animal for maximum vitality and reproductive competence during young adulthood, if allowed to continue, can be detrimental during old age. Androgen receptor-mediated development and growth of the prostate gland is an example of such "antagonistic pleiotropy." The prostate gland is composed of two major groups of cells: the epithelial and stromal. Among the epithelial type, the columnar cells on the luminal surface produce the prostatic secretions, and the basal cells are presumed to serve as progenitors of the columnar cells. Within the stromal cell population, fibroblastic and smooth muscle cells are thought to produce growth factors that support the development and function of the epithelial cells. Both epithelial and stromal cells are dependent on androgens. In this study, we have examined age-dependent expression of the androgen receptor gene in the prostatic tissues of rats and dogs. Unlike the rat, in which the prostatic growth ceases after sexual maturation, the dog prostate continues to grow during aging. Similar to the dog, the antagonistic pleiotropy of the prostatic growth in the human causes the pathological condition of benign prostatic hyperplasia (BPH), the major health problem in old men. Quantitation of the androgen receptor (AR) mRNA in the total prostate extracts from young and old animals by the reverse transcriptase-polymerase chain reaction (RT-PCR) method showed about a 30% decline in AR mRNA in the 24-month-old rat prostate, as compared to the prostate of 3-month-old young adult animals. However, no significant difference in AR mRNA contents between 1-year-old and 10-year-old dog prostates was observed. In situ immunostaining for the androgen receptor protein revealed that in the case of rat, developmental maturation during the first month of life is associated with an increase in AR immunoreactivity in the luminal columnar epithelium, with a concomitant loss of immunoreactivity in the basal cells. Furthermore, with aging, there was a marked increase in the proportion of AR-negative basal cells in comparison to luminal columnar cells. Surprisingly, in both young adult (approximately 1-year-old) and old (approximately 10-year-old) dogs, most of the AR immunoreactivity was localized in the fibroblastic stromal cells rather than in the epithelial cells. Based on these observations and the existing literature, we propose that normally, in most mammalian species, an age-dependent decline in the conversion of basal to columnar epithelial cells after sexual maturation serves as a stop signal for the prostate growth. However, in certain species, such as the dog, robust AR expression in the stromal cells overrides this regulatory blockage and leads to prostatic hyperplasia in old age.

The evolutionary tangle of aging, sex, and reproduction and an experimental approach to its molecular dissection.

Exchange of genetic materials by two individual members of the same species is considered to be the origin of primitive sex. During evolution, this primitive form of molecular sex has been transformed into a complex biological function involving specialized sexual structures and multiple hormonal interactions. Development and maintenance of these reproductive structures are also dependent on hormones and hormone receptors. Furthermore, reproductive specialization in higher forms of life has led to customized species-specific rates of aging and life-span potentials that are commensurate with the reproductive needs of the particular type of organism. Because of this reproductive imposition on aging of the organism, temporal regulation of the hormone response is a significant component of the genetics of aging. We have observed a marked age-dependent alteration in the hepatic expression of the rat androgen receptor (rAR) gene. Among the large number of transcription factors that control the rAR gene, at least three appear to participate in its age-dependent regulation. Two of these are positively acting and yet/to be characterized transcription factors, while the third is a negative regulator the nuclear factor kappa B (NF-kappa B). NF-kappa B is the major trans-regulator for genes involved in the immune response, inflammation, and oxidative stress. Involvement of NF-kB in the modulation of both oxidative stress and sex function provides the first example of a common molecular link between sex and aging.

Regulation of androgen action by receptor gene inhibition.

Regulated functions of hormonal agents play a critical role in health and disease. Target cell responsiveness to a hormonal signal is a product of both cellular concentrations of the hormone ligand and the corresponding receptor protein. The major thrust of the drug design for treatment of endocrine-related problems, so far, has been directed to ligand derivatives. In certain cases, receptor regulation through antigene technology has much to offer with improvements in both target cell and hormonal specificity. Three different antigene approaches are currently being explored. The first approach is to inhibit the expression of the receptor gene by disrupting the DNA protein interaction at critical cis-elements by short triple helix-forming oligonucleotides. The second approach is to sequester and inactivate the receptor mRNA by the antisense mRNA produced in the target tissue directed by a heterologous tissue-specific promoter. The third approach is the tissue-specific expression of a catalytic ribozyme that binds to the specific receptor mRNA and selectively degrades it before its translation into the protein. In this study, we have characterized the promoter of the rat androgen receptor, and by progressive deletion from its 5' end have identified two critical cis-regulatory elements, one at the -960 to -940 region and the other at the -554 to -574 positions. The former is an activator while the latter is an inhibitor domain. The inhibitory domain is the binding site for the nuclear factor kappa B (NF-kappa B) and more specifically, the p50/p50 homodimer of this transcription factor family. We have also provided correlative data to show that under normal physiological conditions, the NF-kappa B functions as an antiandrogen during the age-dependent desensitization of the liver. In addition to the naturally functioning antiandrogenic influence of NF-kappa B, we have designed an artificial antiandrogenic agent, a triplex-forming oligonucleotide (TFO) directed to the -960/-940 activator domain of the rat androgen receptor gene promoter. This oligonucleotide at a TFO-to-promoter ratio of 500 is able to cause about 60% inhibition of rAR promoter function in transfected COS-1 cells. These results clearly demonstrate the feasibility of the antigene approach for effective inhibition of steroid hormone action.

Cloning and sequence analysis of a novel member of the single-stranded DNA binding protein family.

A single-stranded DNA binding protein (SSB) was isolated which has unusual amino acid residues at sites previously shown to be highly conserved and critical for DNA binding. Sequence analysis suggested that these residues are characteristic of a novel class of SSBs from Gram-positive bacteria.

A novel regulatory element associated with age-dependent expression of the rat androgen receptor gene.

A large body of evidence indicates that the genetic program of aging has co-evolved with the sexual mode of reproduction (Partridge, L., and Barton, N. H. (1993) Nature 362, 305-311). Age-dependent changes in target cell sensitivity to reproductive hormones can be considered part of this evolutionary linkage. Here we describe a novel regulatory element in the rat androgen receptor (AR) gene promoter associated with its age-dependent expression in the liver. This element consists of two (19 and 25 base pairs) contiguous sites, one specifically binding an Age-dependent Factor (ADF) and the other an Associated Factor (AF). Both deletion and point mutations of the ADF site result in about a 5-fold decline in the AR promoter function. Unlike AF, which is relatively tissue specific, ADF appears to be ubiquitous. The ubiquitous and evolutionarily conserved nature of ADF suggests a fundamental role of this novel transcription factor in programmed gene expression.

Analysis of nuclear proteins interacting with a wheat alpha/beta-gliadin seed storage protein gene.

The promoter region (-524 to -46) of the wheat alpha/beta-gliadin seed storage protein gene was analyzed for interactions with nuclear proteins from developing wheat seeds. Six complexes were detected within the first 165 bp upstream of the transcriptional start site. One of the proteins was a non-sequence specific AT-binding protein. The remaining five proteins bound in a sequence specific manner. One (CABP) mapped to a conserved CA-rich element at -134 to -112 while another (PalBP) mapped to an adjacent, palindromic sequence at -112 to -106. Three proteins (CTBPs 1-3) formed complexes at two, independent homologous sites. The activities of four of the binding proteins, CTBPs 1-3 and CABP, exhibited similar patterns of expression during seed development: they first appeared at early to mid stages, reached a maximum at mid stage and subsequently decreased, paralleling the pattern of gliadin mRNA accumulation. The non-specific AT-binding protein was detected at relatively high levels only at mid development. PalBP activity, on the other hand, first appeared at mid stage and was present at a constant level throughout later stages of development. The results suggest that the binding proteins may regulate gliadin expression in an antagonistic manner.

The influence of ribosome-binding-site elements on translational efficiency in Bacillus subtilis and Escherichia coli in vivo.

A method is described to determine simultaneously the effect of any changes in the ribosome-binding site (RBS) of mRNA on translational efficiency in Bacillus subtilis and Escherichia coli in vivo. The approach was used to analyse systematically the influence of spacing between the Shine-Dalgarno sequence and the initiation codon, the three different initiation codons, and RBS secondary structure on translational yields in the two organisms. Both B. subtilis and E. coli exhibited similar spacing optima of 7-9 nucleotides. However, B. subtilis translated messages with spacings shorter than optimal much less efficiently than E. coli. In both organisms, AUG was the preferred initiation codon by two- to threefold. In E. coli GUG was slightly better than UUG while in B. subtilis UUG was better than GUG. The degree of emphasis placed on initiation codon type, as measured by translational yield, was dependent on the strength of the Shine-Dalgarno interaction in both organisms. B. subtilis was also much less able to tolerate secondary structure in the RBS than E. coli. While significant differences were found between the two organisms in the effect of specific RBS elements on translation, other mRNA components in addition to those elements tested appear to be responsible, in part, for translational species specificity. The approach described provides a rapid and systematic means of elucidating such additional determinants.

Analysis of an mRNA exhibiting anomalous translational specificity.

Gene 6 mRNA of Bacillus subtilis phage phi 29 is inefficiently translated under standard in vitro conditions by Escherichia coli, while it is efficiently translated by the in vitro system derived from B. subtilis. This is a rare example of the inability of E. coli to translate mRNA translated by B. subtilis. The ionic condition in the translation systems was the key component in the differential recognition of the gene 6 message by E. coli and B. subtilis ribosomes. Its translation by E. coli ribosomes was preferentially inhibited by moderate levels of KCl, while its translation by B. subtilis ribosomes was unaffected by these concentrations of salt. This preferential inhibition with E. coli ribosomes was observed in vitro as well as in vivo. While not influencing the general phenomenon of preferential inhibition, anion-specific effects were observed in overall protein synthesis. Glutamate and acetate promoted efficient synthesis over a broad range of concentrations, whereas chloride was inhibitory at all concentrations tested.