Search

The hammerhead ribozyme is an intriguing RNA molecule with the ability to serve as a catalyst to cleave sequence-specifically RNA molecules in an intermolecular reaction. Preferentially Mg²⁺ is required for optimal activity by inducing the catalytically competent conformation and by possibly acting as an acid–base catalyst. Even though the three-dimensional structure has been elucidated details of the structure–function relationship and of the mechanism remain unanswered. The hammerhead ribozyme has stimulated the concept of the sequence-specific cleavage of mRNAs intracellularly and thus to inhibit gene expression by preventing translation. This represents an area of considerable interest as it has the potential for the development of drugs. © 2001 John Wiley & Sons, Inc. Biopolymers (Nucleic Acid Sci) 52: 147–154, 1999

H3.3 is a replacement histone subtype that is encoded by two replication-independent genes termed H3.3A and H3.3B, respectively. We have isolated a full-size H3.3 cDNA clone from an oligo(dT)-primed human testicular cDNA library. Subsequently, the corresponding gene was isolated from a human cosmid library and was identified as the H3.3B gene. It was the only histone gene on this 42-kb cosmid clone. The gene structure shows characteristic features of an H3.3 gene. First, it contains an intron of about 0.5 kb in the 5′ untranslated region and two smaller introns within the coding gene portion. Second, no histone gene-specific dyad symmetry element was found in the 3′ untranslated region, but three putative polyadenylation signals were detected downstream of the gene. The corresponding transcripts were detected by Northern blot analysis using poly(A)+RNA from testis and from the HEK293 tumor cell line. The newly discovered human H3.3B gene (HGMW-approved symbol H3F3B) was mapped by fluorescencein situhybridization to the telomeric region of chromosome 17 (17q25). This localization of the H3.3B gene and its solitary arrangement contrast with the majority of the replication-dependent histone genes, which form a large cluster on chromosome 6 and a second cluster on chromosome 1.

The histone family of proteins is subdivided into two major groups: the main type histones, which are synthesized in coordination with DNA replication during the S-phase of the cell cycle, and the replacement histones, which can be synthesized in the absence of DNA replication substituting main type histone isoforms. Accumulation of replacement histone variants has been observed in several terminally differentiated tissues that have stopped cell division. The replacement subtype of the H3 class is termed H3.3. This protein is encoded by two different genes (H3.3A and H3.3B) that both code for the same amino acid sequence, but differ in nucleotide sequences and gene organization. This has been shown for human and avian H3.3A and H3.3B genes and for a murine H3.3B cDNA. In an attempt to define patterns of replacement histone H3.3 gene expression during male germ cell differentiation, we have constructed mouse testicular cDNA libraries and have isolated cDNAs corresponding to the murine H3.3A and H3.3B genes. Using probes specific for these two different genes we show by RNase protection analysis and by nonradioactive in situ hybridization with testis sections that H3.3A mRNA is present in pre- and postmeiotic cells, whereas expression of the H3.3B gene is essentially restricted to cells of the meiotic prophase.