The signal for glucose repression of the lactose-galactose regulon is amplified through subtle modulation of transcription of the Kluyveromyces lactis Kl-GAL4 activator gene.

Induction of the lactose-galactose regulon is strongly repressed by glucose in some but not all strains of Kluyveromyces lactis. We show here that in strongly repressed strains, two to three times less Kl-GAL4 mRNA is synthesized and that expression of structural genes in the regulon such as LAC4, the structural gene for beta-galactosidase, is down regulated 40-fold or more. Comparative analysis of strains having a strong or weak repression phenotype revealed a two-base difference in the promoter of the Kl-GAL4 (also called LAC9) positive regulatory gene. This two-base difference is responsible for the strong versus the weak repression phenotype. The two base changes are symmetrically located in a DNA sequence having partial twofold rotational symmetry (14 of 21 bases). We hypothesize that this region functions as a sensitive regulatory switch, an upstream repressor sequence (URS). According to our model, the presence of glucose in the culture medium signals, by an unidentified pathway, a repressor protein to bind the URS. Binding reduces transcription of the Kl-GAL4 gene so that the concentration of the Kl-GAL4 protein falls below the level needed for induction of LAC4 and other genes in the regulon. For strains showing weak glucose repression, we hypothesize that the two base changes in the URS reduce repressor binding so that the regulon is not repressed. Our results illustrate an important principle of genetic regulation: a small (2- to 3-fold) change in the concentration of a regulatory protein can produce a large (40-fold or greater) change in expression of structural genes. This mechanism of signal amplification could play a role in many biological phenomena that require regulated transcription.

Characterization of the 5'-flanking region of the human tartrate-resistant acid phosphatase (TRAP) gene.

Tartrate-resistant acid phosphatase (TRAP) is expressed at high levels in osteoclasts and may play an important role in the bone resorptive process. However, factors regulating human TRAP gene expression have not been clearly defined. Therefore, we isolated a genomic clone (CL-9) for TRAP containing a 14-kb insert. A restriction map was generated for this insert, and a 2.6-kb ApaI fragment containing the 5'-flanking region was subcloned. Sequence analysis of this fragment revealed the presence of candidate transcription factor-binding sequences for H-APF-1, SP1, GATA2, and the c-Myc proto-oncogene. PCR analysis of RNA isolated from human osteoclastomas and pagetic bone revealed a 276-bp intron at -1 bp to -276 bp relative to the ATG and a transcript originating from this intron. Rapid amplification of the 5' end of the human TRAP mRNA by PCR indicated the presence of a 93-bp untranslated region 5' from the intron. Promoter activity was detected in the DNA fragment from +1 bp to -1903 bp relative to the ATG initiation codon, which drove the transient expression of a luciferase reporter gene when transfected into HRE H9 rabbit endometrial cells. Comparison of the human TRAP 5'-flanking region with mouse TRAP and uteroferrin revealed 41% and 47% homology, respectively. This suggests that regulation of human TRAP gene expression may differ from that for the murine TRAP gene.

XAP2, a novel hepatitis B virus X-associated protein that inhibits X transactivation.

The hepatitis B virus X protein is a promiscuous transcriptional transactivator. Transactivation by the X protein is most likely mediated through binding to different cellular factors. Using the yeast two-hybrid method, we have isolated a clone that encodes a novel X-associated cellular protein: XAP2. X and XAP2 interactions also occur in vitro. Antiserum raised against XAP2 recognizes a cytoplasmic protein with an apparent molecular mass of 36 kDa. The interaction between X and XAP2 requires a small region on X containing amino acids 13-26. From Northern blot analyses, XAP2 is ubiquitously expressed in both liver-derived and non-liver-derived cell lines as well as in normal non-liver tissues. In contrast, XAP2 is expressed in very low level in the normal human liver. In transfection assays, overexpression of XAP2 abolishes transactivation by the X protein. Based on these results, we suggest that XAP2 is an important cellular negative regulator of the X protein, and that X-XAP2 interaction may play a role in HBV pathology.

The hepatitis B virus X-associated protein, XAP3, is a protein kinase C-binding protein.

The hepatitis B virus X protein induces transcriptional activation of a wide variety of viral and cellular genes. In addition to its ability to interact directly with many nuclear transcription factors, several reports indicate that the X protein stimulates different cytoplasmic kinase signal cascades. Using the yeast two-hybrid screen, we have isolated a clone designated X-associated protein 3 (XAP3) that encodes a human homolog of the rat protein kinase C-binding protein. One of the activation domains of X (amino acids 90-122) is required for binding to XAP3, while the NH2-terminal part of XAP3 is necessary for binding to X. Both X and XAP3 bound specifically to the eta PKC isoenzyme synthesized in rabbit reticulocyte lysates. Overexpression of XAP3 enhanced X transactivation activity. These results support earlier findings that one of the mechanisms of transactivation by X is through involvement with the cellular protein kinase C pathway.