Efficient and specific ribozyme-mediated reduction of bovine alpha-lactalbumin expression in double transgenic mice.

Transgenic mice carrying a bovine alpha-lactalbumin (alpha-lac) specific ribozyme gene under the transcriptional control of the mouse mammary tumor virus long terminal repeat were generated and cross-bred with animals that highly express a bovine alpha-lac transgene (0.4 mg of alpha-lac/ml(-1) of milk). The ribozyme contains the hammerhead catalytic domain, flanked by 12-nt sequences complementary to the 3' untranslated region of bovine alpha-lac transcript. High-level expression of the ribozyme gene was detected by Northern blot analysis in the mammary gland of 7-8 day lactating transgenic mice, from 3 of 12 lines analyzed. Heterozygous expression of the ribozyme resulted in a reduction in the levels of the target mRNA to 78, 58, and 50% of that observed in the nonribozyme transgenic littermate controls for three independent lines. The ribozyme-mediated reduction in the levels of the bovine protein paralleled that observed for the mRNA, and was positively correlated with the level of expression of the ribozyme. In nonribozyme expressing transgenic mice, the level of bovine alpha-lac mRNA and protein was not affected. The specificity of this activity is demonstrated by the absence of a reduction in the levels of the endogenous murine alpha-lac mRNA or protein. These results demonstrate the feasibility of ribozyme-mediated down-regulation of highly-expressed transcripts in transgenic animals.

Structure and function in rhodopsin: correct folding and misfolding in two point mutants in the intradiscal domain of rhodopsin identified in retinitis pigmentosa.

The rhodopsin mutants P23H and G188R, identified in autosomal dominant retinitis pigmentosa (ADRP), and the site-specific mutants D190A and DeltaY191-Y192 were expressed in COS cells from synthetic mutant opsin genes containing these mutations. The proteins expressed from P23H and D190A partially regenerated the rhodopsin chromophore with 11-cis-retinal and were mixtures of the correctly folded (retinal-binding) and misfolded (non-retinal-binding) opsins. The mixtures were separated into pure, correctly folded mutant rhodopsins and misfolded opsins. The proteins expressed from the ADRP mutant G188R and the mutant DeltaY191-Y192 were composed of totally misfolded non-retinal-binding opsins. Far-UV CD spectra showed that the correctly folded mutant rhodopsins had helical content similar to that of the wild-type rhodopsin, whereas the misfolded opsins had helical content 50-70% of the wild type. The near-UV CD spectra of the misfolded mutant proteins lack the characteristic band pattern seen in the wild-type opsin, indicative of a different tertiary structure. Further, whereas the folded mutant rhodopsins were essentially resistant to trypsin digestion, the misfolded opsins were degraded to small fragments under the same conditions. Therefore, the misfolded opsins appear to be less compact in their structures than the correctly folded forms. We suggest that most, if not all, of the point mutations in the intradiscal domain identified in ADRP cause partial or complete misfolding of rhodopsin.

Sequence-specific DNA-binding dominated by dehydration.

Fluorescence spectroscopy and isothermal titration calorimetry were used to study the thermodynamics of binding of the glucocorticoid receptor DNA-binding domain to four different, but similar, DNA-binding sites. The binding sites are two naturally occurring sites that differ in the composition of one base pair, i.e., an A-T to G-C mutation, and two sites containing chemical intermediates of these base pairs. The calorimetrically determined heat capacity change (Delta C(p)o(obs)) for glucocorticoid receptor DNA-binding domain binding agrees with that calculated for dehydration of solvent-accessible surface areas. A dominating effect of dehydration or solvent reorganization on the thermodynamics is also consistent with an observed linear relationship between observed enthalpy change (Delta Ho(obs)) and observed entropy change (Delta So(obs)) with a slope close to the experimental temperature. Comparisons with structural data allow us to rationalize individual differences between Delta Ho(obs) (and Delta So(obs)) for the four complexes. For instance, we find that the removal of a methyl group at the DNA-protein interface is enthalpically favorable but entropically unfavorable, which is consistent with a replacement by an ordered water molecule.

POU domain factor Brn-3b is required for the development of a large set of retinal ganglion cells.

The three members of the Brn-3 family of POU domain transcription factors are found in highly restricted sets of central nervous system neurons. Within the retina, these factors are present only within subsets of ganglion cells. We show here that in the developing mouse retina, Brn-3b protein is first observed in presumptive ganglion cell precursors as they begin to migrate from the zone of dividing neuroblasts to the future ganglion cell layer, and that targeted disruption of the Brn-3b gene leads in the homozygous state to a selective loss of 70% of retinal ganglion cells. In Brn-3b (-/-) mice other neurons within the retina and brain are minimally or not at all affected. These experiments indicate that Brn-3b plays an essential role in the development of specific ganglion cell types.

Modulation of promoter occupancy by cooperative DNA binding and activation-domain function is a major determinant of transcriptional regulation by activators in vivo.

Binding of transcriptional activators to a promoter is a prerequisite process in transcriptional activation. It is well established that the efficiency of activator binding to a promoter is determined by the affinity of direct interactions between the DNA-binding domain of an activator and its specific target sequences. However, I describe here that activator binding to a promoter is augmented in vivo by the effects of two other determinants that have not been generally appreciated: (i) the number of activator binding sites present in a promoter and (ii) the potency of activation domains of activators. Multiple sites within a promoter can cooperatively recruit cognate factors regardless of whether they contain an effective activation domain. This cooperativity can result in the synergistic activation of transcription. The second effect is the enhancement of activator binding to a promoter by the presence of activation domains. In this case, activation domains are not simply tethered to the promoter by the DNA-binding domain but instead assist the DNA-binding domain being tethered onto the promoter. This effect of activation domains on DNA binding is instrumental in determining how potent activators can induce steep transcriptional increases at low concentrations.

Amino-terminal protein-protein interaction motif (POZ-domain) is responsible for activities of the promyelocytic leukemia zinc finger-retinoic acid receptor-alpha fusion protein.

Promyelocytic leukemia zinc finger-retinoic acid receptor a (PLZF-RARalpha), a fusion receptor generated as a result of a variant t(11;17) chromosomal translocation that occurs in a small subset of acute promyelocytic leukemia (APL) patients, has been shown to display a dominant-negative effect against the wild-type RARalpha/retinoid X receptor alpha (RXRalpha). We now show that its N-terminal region (called the POZ-domain), which mediates protein-protein interaction as well as specific nuclear localization of the wild-type PLZF and chimeric PLZF-RARalpha proteins, is primarily responsible for this activity. To further investigate the mechanisms of PLZF-RARalpha action, we have also studied its ligand-receptor, protein-protein, and protein-DNA interaction properties and compared them with those of the promyelocytic leukemia gene (PML)-RARalpha, which is expressed in the majority of APLs as a result of t(15;17) translocation. PLZF-RARalpha and PML-RARalpha have essentially the same ligand-binding affinities and can bind in vitro to retinoic acid response elements (RAREs) as homodimers or heterodimers with RXRalpha. PLZF-RARalpha homodimerization and heterodimerization with RXRalpha were primarily mediated by the POZ-domain and RARalpha sequence, respectively. Despite having identical RARalpha sequences, PLZF-RARalpha and PML-RARalpha homodimers recognized with different affinities distinct RAREs. Furthermore, PLZF-RARalpha could heterodimerize in vitro with the wild-type PLZF, suggesting that it may play a role in leukemogenesis by antagonizing actions of not only the retinoid receptors but also the wild-type PLZF and possibly other POZ-domain-containing regulators. These different protein-protein interactions and the target gene specificities of PLZF-RARalpha and PML-RARalpha may underlie, at least in part, the apparent resistance of APL with t(11;17) to differentiation effects of all-trans-retinoic acid.

Vascular endothelial growth factor-related protein: a ligand and specific activator of the tyrosine kinase receptor Flt4.

The tyrosine kinases Flt4, Flt1, and Flk1 (or KDR) constitute a family of endothelial cell-specific receptors with seven immunoglobulin-like domains and a split kinase domain. Flt1 and Flk1 have been shown to play key roles in vascular development; these two receptors bind and are activated by vascular endothelial growth factor (VEGF). No ligand has been identified for Flt4, whose expression becomes restricted during development to the lymphatic endothelium. We have identified cDNA clones from a human glioma cell line that encode a secreted protein with 32% amino acid identity to VEGF. This protein, designated VEGF-related protein (VRP), specifically binds to the extracellular domain of Flt4, stimulates the tyrosine phosphorylation of Flt4 expressed in mammalian cells, and promotes the mitogenesis of human lung endothelial cells. VRP fails to bind appreciably to the extracellular domain of Flt1 or Flk1. The protein contains a C-terminal, cysteine-rich region of about 180 amino acids that is not found in VEGF. A 2.4-kb VRP mRNA is found in several human tissues including adult heart, placenta, ovary, and small intestine and in fetal lung and kidney.

E2F1 and E1A(12S) have a homologous activation domain regulated by RB and CBP.

The E2F1 transcription factor has a well-characterized activation domain at its C terminus and the E1A protein has a recently defined activation domain at its N terminus. Here we show that these activation domains are highly related in sequence. The sequence homology reflects, at least partly, the conservation of common binding sites for the RB and CBP/p300 proteins, which are preserved in the same relative order along E2F1 and E1A. Furthermore, the interaction of RB and CBP with these two activation domains results in the same functional consequences: RB represses both activation domains, whereas CBP stimulates them. We conclude that the activation domains of E1A(12s) and E2F1 belong to a novel functional class, characterized by specific protein binding sites. The implication of this conservation with respect to E1A-induced stimulation of E2F activity is discussed.