Formation of prostatic buds from the urogenital sinus (UGS) to initiate prostate development requires localized action of several morphogenetic factors. This report reveals all-trans-retinoic acid (RA) to be a powerful inducer of mouse prostatic budding that is associated with reciprocal changes in expression of two regulators of budding: sonic hedgehog (Shh) and bone morphogenetic protein 4 (Bmp4). Localization of retinoid signaling and expression of RA synthesis, metabolism, and receptor genes in the UGS on embryonic days 14.5-17.5 implicate RA in the mechanism of bud initiation. In UGS organ culture, RA increased prostatic budding, increased Shh expression, and decreased Bmp4. Prostatic budding was stimulated in the absence of RA by recombinant SHH, by blocking BMP4 signaling with NOGGIN, or by combined treatment with SHH and NOGGIN in UGS organ culture media. These observations suggest that reciprocal changes in hedgehog and BMP signaling by RA may regulate bud initiation. Developmental Dynamics 237:1321-1333, 2008.

Nucleotide variations in the human genome, such as single‐nucleotide polymorphisms, have been researched more intensively since it became apparent that these deviations are linked to various diseases and also several side effects of drugs. The investigation of genomic DNA in the laboratory requires routine methods that are time‐, labour‐, and cost‐effective. These criteria are fulfilled by so‐called closed‐tube methods, which are applied directly to isolated genomic DNA without any preamplification.

DNA polymerase selectivity often varies significantly depending on the DNA polymerase. The origin of this varying error propensity is elusive. It is assumed that DNA polymerases form nucleotide binding pockets that differ in properties such as shape and tightness. We tested this prediction and studied HIV-1 RT by employment of size-augmented nucleotides and site-directed mutagenesis of the enzyme. New valuable insights into the mechanism of DNA polymerase fidelity were obtained. The presented study provides experimental evidence that variations of steric constraints within the nucleotide binding pocket of at least two DNA polymerases cause variations in nucleotide incorporation selectivity. Thus, our results support the concept of active site tightness as a causative in differential fidelity among DNA polymerases.

The selectivity of DNA polymerases for processing the canonical nucleotide and DNA substrate in favor of the noncanonical ones is the key to the integrity of the genome of every living species and to many biotechnological applications. The inborn ability of most DNA polymerases to abort efficient extension of mismatched DNA substrates adds to the overall DNA polymerase selectivity. DNA polymerases have been grouped into families according to their sequence. Within family A DNA polymerases, six motifs that come into contact with the substrates and form the active site have been discovered to be evolutionary highly conserved. Here we present results obtained from amino acid randomization within one motif, motif C, of thermostable Thermus aquaticus DNA polymerase. We have identified several distinct mutation patterns that increase the selectivity of mismatch extension. These results might lead to direct applications such as allele-specific PCR, as demonstrated by real-time PCR experiments and add to our understanding of DNA polymerase selectivity.

To examine the concept of polymerase active site tightness as a criteria for DNA polymerase fidelity, we performed pre-steady-state single nucleotide incorporation kinetic analyses with sugar modified thymidine 5-triphosphate (TTP) analogues and human immunodeficiency virus (HIV-1) reverse transcriptase (RT). The employed TTP analogues (T R TP) are modified at the 4-position of the sugar moiety with alkyl groups, gradually expanding their steric demand. Introduction of a methyl group reduces the maximum rate of nucleotide incorporation by about 200-fold for RT WT The intrinsic error frequencies of DNA polymerases are typically in the range of 10 Ϫ3 to 10 Ϫ5 per base replicated (1-3). Most eukaryotic DNA polymerases show higher fidelity, whereas virally encoded polymerases are more error-prone. Recently, a new class of so called bypass polymerases have been discovered (4 -8). These polymerases are thought to have certain functions in DNA repair rather than replication and show error rates as high as 1 per 22 bases (9). Thus, depending on their function, polymerases show different degrees of selectivity for the nucleotide substrate. For example, it is conceivable that a viral polymerase has lower fidelity than a cellular polymerase, enabling the virus to escape the host immune system response by an increased mutation rate. Although our knowledge about polymerases has grown substantially in the past few years, the mechanistic details for this varied substrate selectivity are not fully understood (10 -12). It is generally accepted that Watson-Crick hydrogen bonding by itself does not account for the observed selectivity. Several additional factors have been discussed to be involved in correct nucleotide recognition (2, 13-15). Among these factors are exclusion of water from the active site of the enzyme, base stacking, solvation, minor groove scanning, and steric constraints within the nucleotide binding pocket (16). It remains to be determined to what extent these factors contribute to polymerase fidelity.Here we used the human immunodeficiency virus (HIV) 1 reverse transcriptase (RT) as a model system to examine the concept of active site tightness and substrate fit as a major determinant of nucleotide selectivity. The HIV-1 enzyme shows a moderate fidelity of about 10 Ϫ4 (17, 18). Interestingly, the mutation M184V, which provides high level resistance to the drug Lamivudine (3TC), has been shown to result in increased fidelity (19 -22). Structural investigations indicate that a ␤-methyl side chain present in valine contacts the sugar ring of the incoming triphosphate, leading to steric hindrance (23-25). This might indicate that small changes of the geometry of the nucleotide binding pocket indeed affect fidelity. If this is the case, modifications of the nucleotide at this position should be felt by the enzyme leading to enhanced discrimination. As a steric probe, we used sugar modified thymidine 5Ј-triphosphate (TTP) analogues (26). In these TTP analogues (T R TP) the 4Ј-hydrogen position of the sugar is subs...