In vivo selection of single-chain antibodies using a yeast two-hybrid system.

The current methodology for screening libraries of single-chain fragments of immunoglobulin variable domains (sFvs) utilizes bacterial phage systems. We have developed a unique in vivo selection protocol combining a modified yeast two-hybrid assay with a novel prey vector expressing sFvs. The viability of the system is demonstrated with the screen of a sFv library cloned into a yeast two-hybrid prey vector for molecules that target the bait ATF-2, a member of the CREB/ATF family of transcriptional regulatory proteins. The isolated sFv was capable of recognizing ATF-2 in vitro on Western blots and in vivo in mammalian cells.

Characterization of the equine glycoprotein hormone alpha-subunit gene reveals divergence in the mechanism of pituitary and placental expression.

The equine glycoprotein hormone alpha-subunit gene is expressed in both pituitary and placenta, unlike that of all other nonprimate mammals studied, in which expression is limited to pituitary. Previous studies of the 5'-flanking region of the equine alpha-subunit promoter have revealed unique characteristics as well as similarities with the human alpha-subunit promoter, which demonstrates a similar pattern of tissue-specific expression. We have cloned and sequenced the equine alpha-subunit gene and have used tissue culture systems and transgenic mice to characterize its expression. Unlike the human promoter, the cloned equine alpha-subunit promoter failed to direct trophoblast-specific expression in either tissue culture or transgenic mouse models, suggesting an entirely different mechanism for expression. In contrast, the equine alpha-subunit promoter was able to direct gonadotroph expression in both tissue culture and transgenic mouse models. In alphaT3-1 cells, 550 base pair (bp) was sufficient for expression. This expression involves promoter elements identified in other species as playing a role in gonadotroph expression, but mutation of these elements reveals differences in their relative contributions to promoter activity. In mice, 2800 bp of 5'-flanking sequence allowed specific expression in gonadotrophs but not in thyrotrophs or placenta. The pattern of estrogen regulation observed in transgenic mice matched neither the repression that has been observed with human and bovine alpha-subunit promoters in transgenic mice nor the stimulation in mRNA levels reported in mares, suggesting a unique mechanism that is not recapitulated in the transgenic model. Thus the equine alpha-subunit promoter uses a combination of conserved and unique features of gene regulation to direct its pattern of tissue-specific expression.

Multiple characteristics of a pentameric regulatory array endow the human alpha-subunit glycoprotein hormone promoter with trophoblast specificity and maximal activity.

Trophoblast-specific expression of the human alpha-subunit glycoprotein hormone gene requires a tightly linked array of five different regulatory elements [trophoblast-specific element (TSE), alpha-activating element (alphaACT), a tandem cAMP response element (CRE), junctional regulatory element (JRE), and a CCAAT box]. We examined their contextual contributions to trophoblast-specific expression by using transfection assays to evaluate activity of systematic block replacement mutations made within the 1500-bp 5'-flanking region of the human alpha-subunit gene. While all five elements were required for full activity, only the TSE and JRE displayed trophoblast specificity. Interestingly, the TSE-binding protein has limited tissue distribution whereas a JRE-binding protein appears trophoblast specific. Likewise, replacement studies with an AP-1 element that binds heterodimers of jun and fos indicated that this element was incapable of compensating for either the tandem CRE or JRE. This preference for both CRE- and JRE-binding proteins provides another avenue for configuring an alpha-subunit promoter with trophoblast specificity. Additional analysis with a cAMP response element binding protein (CREB)-Gal4 fusion protein further underscored the importance of CREB as well as suggested that transcriptional contributions come from both the DNA-binding domain and transactivation domain of this protein. We also examined the interactive nature of the pentameric array by placing a 15-bp random sequence between each element. Remarkably, only the insertion 3' of the CCAAT box diminished promoter activity. This suggested the absence of direct interactions between the transcriptional factors that bind each element in the array. It also suggested that the CCAAT box is position-dependent relative to the TATA box. This position dependence appeared cell-specific, as it was not manifest in a gonadotrope cell line (alphaT3-1 cells). Thus, the CCAAT box also has tissue-specific characteristics that assist in targeting expression of the alpha-subunit gene to trophoblasts. Together, these data suggest that multiple characteristics of a complex pentameric array of regulatory elements endow the alpha-subunit promoter with trophoblast specificity and maximal activity.

Relationships between computerized measurements of motion of frozen-thawed bull spermatozoa and fertility.

A computerized system (CellSoft, CRYO Resources, Ltd.) was validated using video tapes of frozen-thawed bull spermatozoa diluted in filtered (0.2 micron) egg yolk-citrate extender (8 X 10(6) spermatozoa/ml) and analyzed at 30 frames/sec for the percentage of motile spermatozoa (greater than or equal to 20 microns/sec) and linear velocity of motile spermatozoa. Virtually all motile spermatozoa were detected and debris rarely were classified as immotile spermatozoa if the extender had been filtered. Variation about the mean for percent motile cells was similar when only 12 rather than 20 or 30 frames/field were analyzed. Use of 20 frames/field was adequate to determine the percentage of motile bull spermatozoa. Five mixtures of live and killed spermatozoa were analyzed (four bulls) to evaluate accuracy. Percent motile spermatozoa was correlated (r = 0.97) with the ratio of live:killed spermatozoa. Mean linear velocity of motile spermatozoa was similar for each mixture (P greater than 0.05). To further evaluate accuracy, percent motile spermatozoa was determined by computer and by "track motility" (20 samples; 0 to 63% motile spermatozoa); values were correlated (r = 0.95). The system was precise (CV of 6% based on triplicate analyses of the same samples) and reasonably accurate for evaluating bull sperm motility if the extender had been filtered and 20 to 25 fields (greater than or equal to 200 spermatozoa) were evaluated. Correlations between measurements of sperm motion and fertility were studied using cryopreserved semen from two fertility trials. For the first, 75-day nonreturn rate data for 20 samples of bull semen (10 bulls) were not significantly correlated with evaluations made by CellSoft. For the second fertility trial, the competitive fertility index (a measure of relative fertility) for nine bulls was correlated (r greater than or equal to 0.68; P less than 0.05) with percent motile spermatozoa, linear velocity and straight-line velocity. Multiple correlations based on six characteristics evaluated by CellSoft, at 0 or 1.5 hours, and the competitive fertility index were greater than or equal to 0.94. Based on the latter data, the system may facilitate prediction of the relative fertility of bull spermatozoa.

A microcomputer-photographic method for evaluation of motility and velocity of bull sperm.

A semiautomated computerized system analyzed 35-mm negatives obtained from dark-field microscopy and determined velocity of each sperm and percentage of motile sperm in the sample. Precision and accuracy of the system for percentage of motile sperm were evaluated in comparisons with track motility and videomicrography. Motility was evaluated at four times during processing for cryopreservation or after thawing. Based on the 95% confidence intervals for percentage of motile sperm, computer and track motility evaluations were more precise than videomicrography. Computer and track determinations of the percentage of motile sperm were correlated with the ratio of live to killed sperm in prepared mixtures. The computer system was reasonably precise and accurate. Correlations of spermatozoal velocity or motility with fertility were studied. The competitive fertility index, based on semen from nine bulls, was correlated with the computer-determined percentage of motile sperm at 0 to 1.5 h after thawing, velocity, or the combination of motile sperm and velocity. Such a computerized system may aid in prediction of fertility.