Otto Hagenbüchle

Changes in size and secondary structure of the ribosomal transcription unit during vertebrate evolution

Abstract Ribosomal RNA and precursor ribosomal RNA from at least one representative of each vertebrate class have been analyzed by electron microscopic secondary structure mapping. Reproducible patterns of hairpin loops were found in both 28 S ribosomal and precursor ribosomal RNA, whereas almost all the 18 S ribosomal RNA molecules lack secondary structure under the spreading conditions used. The precursor ribosomal RNA of all species analyzed have a common design. The 28 S ribosomal RNA is located at or near the presumed 5′-end and is separated from the 18 S ribosomal RNA region by the internal spacer region. In addition there is an external spacer region at the 3′-end of all precursor ribosomal RNA molecules. Changes in the length of these spacer regions are mainly responsible for the increase in size of the precursor ribosomal RNA during vertebrate evolution. In cold blooded vertebrates the precursor contains two short spacer regions; in birds the precursor bears a long internal and a short external spacer region, and in mammals it has two long spacer regions. The molecular weights, as determined from the electron micrographs, are 2·6 to 2·8 × 10 6 for the precursor ribosomal RNA of cold blooded vertebrates, 3·7 to 3·9 × 10 6 for the precursor of birds, and 4·2 to 4·7 × 10 6 for the mammalian precursor. Ribosomal RNA and precursor ribosomal RNA of mammals have a higher proportion of secondary structure loops when compared to lower vertebrates. This observation was confirmed by digesting ribosomal RNAs and precursor ribosomal RNAs with single-strandspecific S 1 nuclease in aqueous solution. Analysis of the double-stranded, S 1 -resistant fragments indicates that there is a direct relationship between the hairpin loops seen in the electron microscope and secondary structure in aqueous solution.

Two promoters of different strengths control the transcription of the mouse alpha-amylase gene Amy-1a in the parotid gland and the liver

We show that two promoters of different strengths are involved in the tissue-specific expression of the alpha-amylase gene Amy-1a in the parotid gland and the liver of mouse. The weaker of the two promoters directs the synthesis of mRNA with a liver-type leader sequence. This promoter is active in both tissues. A promoter that is about 30-fold stronger is exclusively active in the parotid, where it directs the synthesis of an mRNA with a parotid-specific leader sequence. Neither the parotid nor the liver promoter is used in tissues that do not contain cytoplasmic alpha-amylase mRNAs, such as brain, kidney, and spleen. Nuclear transcripts that are initiated several kilobases upstream of the parotid cap site are detected in several tissues. They are most abundant in brain, and are apparently not processed into alpha-amylase mRNA.

A single mouse α-amylase gene specifies two different tissue-specific mRNAs

Abstract The α-amylase mRNAs which accumulate in two different tissues of the mouse, the salivary gland and the liver, are identical except for their 5′ nontranslated sequences: the 5′ terminal 158 nucleotides of the major liver α-amylase mRNA are unrelated to the 5′ terminal 47 nucleotides found in its salivary gland counterpart. DNA that specifies the 5′ terminal one-quarter of these mRNAs has been isolated through genomic cloning and sequenced. The initial 161 nucleotides of the liver α-amylase mRNA are specified by DNA sequences that lie 4.5 kb upstream from those for the common body of the two mRNAs. In contrast, the 5′ terminal 50 nucleotides of the salivary gland α-amylase mRNA are found 7.5 kb from sequences that the two mRNAs share in the genome. These cloned DNA sequences occur once per haploid genome, indicating that both the salivary gland and liver α-amylase mRNAs are transcribed from the same gene ( Amyl A ). Since no rearrangement of these DNA sequences can be detected among mouse sperm, salivary gland or liver preparations, gross rearrangement does not account for the tissue-specific pattern of expression observed for Amyl A . Rather, these data indicate that the salivary gland and liver α-amylase mRNAs are differentially transcribed and/or processed from identical DNA sequences in different tissues.

Nuclear targeting of the transcription factor PTF1 is mediated by a protein subunit that does not bind to the PTF1 cognate sequence

The pancreas-specific transcription factor PTF1 is a heterooligomer that exists as two variants, alpha and beta, both of which bind DNA. The nucleus contains exclusively alpha while the cytoplasm contains both forms. Alpha and beta differ in protein composition. Reconstitution of alpha in vitro requires, in addition to the DNA-binding subunits common to both forms, a 75 kd glycosylated protein that apparently does not bind DNA. Here we show that this protein is essential for targeting PTF1 to the nucleus. Upon injection into frog oocytes, alpha is translocated quantitatively to the nucleus while beta remains in the cytoplasm. However, if beta is coinjected with purified 75 kd protein or a particular size fraction of pancreatic mRNA, it can be converted to alpha and imported into the nucleus.

The mouse α-amylase multigene family sequence organization of members expressed in the pancreas, salivary gland and liver☆

The DNAs that specify the α-amylase messenger RNAs found in the pancreas, salivary gland and liver of mouse strain A have been isolated by molecular cloning in phage λ. Amylase clones were studied by mRNA/DNA hybrid analysis in the electron microscope, restriction endonuclease site mapping and DNA sequencing. The Amy-2a gene, which specifies pancreatic α-amylase mRNA, measures 10·1 kb from cap to polyadenylation site and is interrupted by at least 9 intervening sequences. Amy-1a, which specifies both salivary gland and liver α-amylase mRNAs contains at least 10 introns. The distance between the cap and polyadenylation sites used in the salivary gland and the liver measures 22·9 kb and 20 kb, respectively. Introns are located at very similar, if not identical, positions within comparable regions of Amy-1a and Amy-2a. The first intron of Amy-1a, which interrupts sequences specifying 5′ non-translated regions of salivary gland and liver α-amylase mRNAs, has no counterpart in Amy-2a. Some introns exhibit considerable sequence homology, suggesting that Amy-1a and Amy-2a have evolved by duplication from a common split ancestor sequence. Repetitive sequence elements occur in the introns and flanking regions of these genes. Gene titration by quantitative autoradiography reveals only one copy of Amy-1a, but two copies of Amy-2a per haploid mouse genome. In addition to Amy-1a and Amy-2a, several other amylase-like DNA sequences exist in the mouse genome. No gross rearrangements of amylase DNA sequences can be detected between germline DNA and that of various mouse tissues.

Termination of transcription in the mouse α-amylase gene Amy-2a occurs at multiple sites downstream of the polyadenylation site

We have delimited the region of transcription termination in the alpha-amylase gene Amy-2a. Mapping of in vitro elongated nascent transcripts to Amy-2a restriction fragments indicates that transcription terminates in a region between 2.5 and 4 kb downstream of the polyadenylation site. These runoff transcription experiments, combined with S1 nuclease mapping of nuclear transcripts at steady state, suggest that transcription termination occurs at multiple sites.

Expression of mouse Amy-2a alpha-amylase genes is regulated by strong pancreas-specific promoters☆

Three types of Amy-2-related DNA sequences, Amy-2a I, Amy-2a II and Amy-X, exist in the genome of mice of the inbred strain A/J. Amy-2a I and Amy-X are single copy sequences. Amy-2a II occurs as three copies per haploid genome. DNA sequence analysis reveals that both classes of Amy-2a genes specify the same unique pancreatic alpha-amylase mRNA species, since they share common exon sequences. Four independently cloned Amy-2a II isolates were found to be identical in all regions sequenced. This suggests that most, if not all, chromosomal Amy-2a II copies are identical. Amy-X is presumably a pseudogene, since its exon sequences, which are distinct from those of Amy-2a, are not detected in pancreatic alpha-amylase mRNA. We have determined the transcriptional activities of the Amy-2a genes by mapping in vitro elongated nascent transcripts to Amy-2a restriction fragments. Transcription initiation occurs at or close to the cap site. The expression of Amy-2a in vivo is under control of strong promoters, which are active exclusively in the pancreas. The accumulation of alpha-amylase mRNA in cells of the exocrine pancreas is regulated mainly at the transcriptional level. We have searched for pancreatic transcripts of Amy-1a, which specifies both parotid gland and liver-type alpha-amylase mRNAs. Surprisingly, the weak Amy-1a promoter, which directs the synthesis of the mRNA containing the liver-type leader sequence, also is active in the pancreas and, hence, in all alpha-amylase-producing tissues.

Incorporation of [5-3H]uridine into ribonucleotide pools and RNA during thyroxine-induced metamorphosis of Xenopus laevis tadpoles

Incorporation of [3H] uridine into the ribonucleoside triphosphates UTP and CTP, total RNA, and nuclear and cytoplasmic RNA was followed in Xenopus laevis tadpole liver during thyroxine (T4)-induced metamorphosis. Pool sizes of UTP and CTP were found to remain unchanged, although turnover the ribonucleoside triphosphates was found to be greatly stimulated after 4 days of hormone treatment. The time course of labeling of the 40-S pre-rRNA was very similar to that of UTP in both thyreostatic and T4-treated tadpoles, thus reflecting a direct relationship between turnover of the immediate precursors and labeling of RNA. Although a faster depletion of labeled UTP and pre-rRNA (precursor ribosomal RNA) was noted in T4-treated tadpoles, labeled cytoplasmic rRNA continued to accumulate almost linearly for 25 h. In thyreostatic larvae no further increase in labeled cytoplasmic rRNA occurred beyond 4 h of labeling. From these results we conclude that both enhanced transcription and more effective utilization of pre-rRNA are responsible for the net accumulation of rRNA observed on the 4th day of T4-induced metamorphosis.