Peter K. Wellauer

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.

The structural organization of ribosomal DNA in drosophila melanogaster

The genes coding for the large rRNAs (rDNA) were purified from Drosophila melanogaster embryos by two different methods. Purified rDNA was analyzed by gel electrophoresis after digestion with restriction endonucleases and by electron microscopy. Each repeating unit of rDNA consists of a gene region that codes for a transcript which includes the 18S and 28S rRNA sequences plus a nontrascribed spacer. Analysis of rRNA/rDNA hybrids in the electron microscope reveals that two types of repeating units exist in the rDNA from this fly. One of these is characterized by insertions of various lengths within the 28S gene. Insertions range in size from about 0.5 kilo base pairs (kb) to 6.0 kb and occur in distinct size classes which are multiples of 0.5 kb. Figure 10 summarizes the structure of the rDNA repeating unit. Repeats with insertions constitute about two thirds, and repeats without insertions about one third of all repeating units in the sample studied where 86% of the rDNA is derived from the X chromosome nucleolus organizer. Statistical analysis of pairs of of nearest-neighbor repeats demonstrates that repeats with and without insertions are interspersed in the X chromosome nucleolus organizer, and that scrambling is close to that expected for random arrangement. Insertions are the major source of length heterogeneity in this rDNA. A minor length heterogeneity also exists in thie nontranscribed spacer of Drosophila rDNA as demonstrated by heteroduplex mapping of restriction fragments in the electron microscope.

The molecular basis for length heterogeneity in ribosomal DNA from Xenopus laevis

The restriction endonuclease EcoRI cleaves Xenopus laevis ribosomal DNA twice in each repeating unit to yield two classes of fragments. One class is homogeneous in length and contains only gene sequences; the other class is heterogeneous in length and contains all of the non-transcribed spacer and some of the gene regions (see model in the Introduction). Four spacer-containing fragments of different length have been cloned in Escherichia coli. They have been compared by optical melting and by homoduplex and heteroduplex mapping in the electron microscope. The results show that two regions within the nontranscribed spacer account for the length heterogeneity. The region which varies most in length is adjacent to the transcription unit at its 5′ end, while the other variable-length region is near but not adjacent to the 3′ end of the transcription unit. Each of these variable-length spacer regions consists of internally repetitious simple sequences (“subrepeats”), which are probably shorter than 50 base-pairs in length. The length heterogeneity of rDNA repeating units is due to more or less copies of these subrepeats. These two regions of variable length are separated by a constant-length region. Another constant-length DNA region separates the 3′ end of the transcription unit from one of the variable spacer sequences. There is no evidence for subrepeats within these latter two spacer regions. It is proposed that the two variable-length regions within the spacer participate in and perhaps enhance the correction mechanisms which permit parallel evolution of tandem genes.

Tissue-specific expression of mouse α-amylase genes☆

Abstract Nearly full-length complementary DNA copies of amylase messenger RNA from mouse pancreas and salivary gland have been cloned and used to compare the cellular concentration, size and structural relationship of amylase mRNAs from pancreas, salivary gland and liver. We find that amylase mRNA is one of the most abundant mRNAs in the exocrine pancreas (10 5 molecules/cell), while its concentration is lower in the salivary gland (10 4 molecules/cell) and in the liver (10 2 molecules/cell). The lengths of deadenylated amylase mRNAs, isolated from pancreas, salivary gland and liver, are 1·65 × 10 3 , 1·72 × 10 3 and 1·80 × 10 3 bases, respectively. Thermal melting studies of RNA-DNA hybrids indicate that the homology between pancreas amylase mRNA and amylase mRNA from either salivary gland or liver is about 90%; melts of hybrids between salivary amylase cDNA and liver amylase mRNA suggest that little if any sequence heterogeneity occurs within the hybridized region of these two mRNAs. As judged by S 1 nuclease digestion experiments and electron microscopy, hybrids between pancreas cDNA and salivary gland or liver amylase mRNAs exhibit considerable sequence heterogeneity over a stretch of about 150 nucleotides within the protein coding region. The high degree of heterogeneity in this region is reflected in the restriction endonuclease cleavage pattern of cloned cDNA from pancreas and salivary gland amylase mRNA. The results indicate that salivary gland and liver amylase mRNAs are transcribed from identical or very closely related genes which differ from that (those) expressed in the pancreas.

Intermolecular duplexes in heterogeneous nuclear RNA from HeLa cells.

Rapidly sedimenting hnRNA complexes contain regions of stable intermolecular duplex. Disruption of such complexes, as judged by a reduction in sedimentation rate, requires conditions sufficient to denature the duplex regions. Rapidly sedimenting molecules reappear only when the complementary sequences reanneal-that is, the formation of such complexes is dependent upon time and the concentration of homologous RNA. These experiments lead us to the conclusion that rapidly sedimenting hnRNA complexes consist of two or more largely single-stranded RNA molecules held together by short duplex regions. Precisely such structures have been visualized in the electron microscope. Rapidly sedimenting fractions of native nuclear RNA from preparative sucrose gradients consist primarily of large, multi-molecular complexes interconnected by duplex regions averaging 300 base pairs in length. Exposure of the RNA to severely denaturing conditions eliminates such complexes. Reannealing of the RNA reconstitutes complexes which are indistinguishable from those observed in preparations before denaturation.

Secondary structure maps of ribosomal RNA and DNA: I. Processing of Xenopus laevis ribosomal RNA and structure of single-stranded ribosomal DNA

Abstract Precursor and mature ribosomal RNA molecules from Xenopus laevis were examined by electron microscopy. A reproducible arrangement of hairpin loops was observed in these molecules. Maps based on this secondary structure were used to determine the arrangement of sequences in precursor RNA molecules and to identify the position of mature rRNAs within the precursors. A processing scheme was derived in which the 40 S rRNA is cleaved to 38 S RNA, which then yields 34 S plus 18 S RNA. The 34 S RNA is processed to 30 S, and finally to 28 S rRNA. The pathway is analogous to that of L-cell rRNA but differs from HeLa rRNA in that no 20 S rRNA intermediate was found. X. laevis 40 S rRNA ( M r = 2.7 × 10 6 ) is much smaller than HeLa or L-cell 45 8 rRNA ( M r = 4.7 × 10 6 ), but the arrangement of mature rRNA sequences in all precursors is very similar. Experiments with ascites cell 3′-exonuclease show that the 28 S region is located at or close to the 5′-end of the 40 S rRNA. Secondary structure maps were obtained also for single-stranded molecules of ribosomal DNA. The region in the DNA coding for the 40 S rRNA could be identified by its regular structure, which closely resembles that of the RNA. Regions corresponding to the 40 S RNA gene alternate with non-transcribed spacer regions along strands of rDNA. The latter have a large amount of irregular secondary structure and vary in length between different repeating units. A detailed map of the rDNA repeating unit was derived from these experiments. Optical melting studies are presented, showing that rRNAs with a high (G + C) content exhibit significant hypochromicity in the formamide/urea-containing solution that was used for spreading.

X and Y chromosomal ribosomal DNA of Drosophila: comparison of spacers and insertions.

Abstract In Drosophila melanogaster, the genes coding for 18S and 28S ribosomal RNA (rDNA) are clustered at one locus each on the X and the Y chromosomes. We have compared the structure of rDNA at the two loci. The 18S and 28S rRNAs coded by the X and Y chromosomes are very similar and probably identical (Maden and Tartof, 1974). In D. melanogaster, many rDNA repeating units are interrupted in the 28S RNA sequence by a DNA region called the insertion. There are at least two sequence types of insertions. Type 1 insertions include the most abundant 5 kilobase (kb) class and homologous small (0.5 and 1 kb) insertions. Most insertions between 1.5 and 4 kb have no homology to the 5 kb class and are identified as type 2 insertions. In X rDNA, about 49% of all rDNA repeats have type 1 insertions, and another 16% have type 2 insertions. On the Y chromosome, only 16% of all rDNA repeats are interrupted, and most if not all insertions are of type 2. rDNA fragments derived from the X and Y chromosomes have been cloned in E. coli. The homology between the nontranscribed spacers in X and Y rDNA was studied with cloned fragments. Stable heteroduplexes were found which showed that these regions on the two chromosomes are very similar. The evolution of rDNA in D. melanogaster might involve genetic exchange between the X and Y chromosomal clusters with restrictions on the movement of type 1 insertions to the Y chromosome.

Ribosomal DNA in Drosophila melanogaster. I. Isolation and characterization of cloned fragments.

Abstract Fragments of rDNA‡ from Drosophila melanogaster produced by the restriction endonuclease Eco RI were cloned in the form of recombinant plasmids in Escheriehia coli . Maps were prepared showing the location of the coding regions and of several restriction endonuclease sites. Most rDNA repeats have a single Eco RI site in the 18 S gene region. Thus, 19 of 24 recombinant clones contained a full repeat of rDNA. Ten repeats with continuous 28 S genes and repeats containing insertions in the 28 S gene of 0.5, 1 and 5 kb were isolated. The 0.5 and 1 kb insertion sequences are homologous to segments of the 5 kb insertions; because of this homology they are grouped together and identified as type 1 insertions. Four recombinant clones contain an rDNA fragment that corresponds to only a portion of a repeating unit. In these fragments the 28 S gene is interrupted by a sequence which had been cleaved by Eco RI. The interrupting sequences in these clones are not homologous to any portion of type 1 insertions and are therefore classified as type 2. In one of the above clones the 28 S gene is interrupted at an unusual position; such a structure is rare or absent in genomic rDNA from the fly. Another unusual rDNA fragment was isolated as a recombinant molecule. In this fragment the entire 18 S gene and portions of the spacer regions surrounding it are missing from one repeat. A molecule with the same structure has been found in uncloned genomic rDNA by electron microscopic examination of RNA/DNA hybrids.

The arrangement of length heterogeneity in repeating units of amplified and chromosomal ribosomal DNA from Xenopus laevis

Non-transcribed spacer regions of Xenopus laevis ribosomal DNA have been found which vary in length between 1.8 × 106 and 5.5 × 106 daltons. Length variation of rDNA† repeats exists within a single nucleolar organizer. Amplified rDNA contains repeats of the same size classes but often in different abundance than the chromosomal rDNA of the same animal. If a certain repeat length is preferred during amplification in an individual, it is also preferred in siblings with the same chromosomal rDNA composition. Thus, preference for a size class in amplification is inherited. Some animals selectively amplify repeat lengths which are rarely found in their chromosomal rDNA; others amplify their most abundant size class. The intramolecular arrangement of length variability was analyzed by the electron microscopy of heteroduplex molecules. Long single strands from two separate preparations of amplified and chromosomal rDNA each were reannealed with an homogeneous cloned spacer-containing rDNA fragment (CD30), and the size of adjacent heteroduplex regions was determined. The arrangement of length heterogeneity is very different in the two types of rDNA. Most, if not all, tandem repeats along a single molecule of amplified rDNA are equal in length. This observation supports a rolling circle mechanism for amplification. In contrast, between 50% and 68% of adjacent repeats in a given molecule of chromosomal rDNA differ in length. For one of the chromosomal rDNA preparations analyzed, the frequency of non-identical nearest-neighbors is compatible with random scrambling of repeats of different lengths. This result bears on the mechanism by which tandem genes evolve. It rules out sudden correction mechanisms of tandem genes such as the “master-slave” or certain “expansion-contraction” models, which predict that tandem genes will be identical.

Secondary structure maps of ribosomal RNA: II. Processing of mouse L-cell ribosomal RNA and variations in the processing pathway☆

Secondary structure mapping in the electron microscope was applied to ribosomal RNA and precusor ribosomal RNA molecules isolated from nucleoli and the cytoplasm of mouse L-cells. Highly reproducible loop patterns were observed in these molecules. The polarity of L-cell rRNA was determined by partial digestion with 3′-exonuclease. The 28 S region is located at the 5′-end of the 45 S rRNA precursor. Together with earlier experiments on labeling kinetics, these observations established a processing pathway for L-cell rRNA. The 45 S rRNA precursor is cleaved at the 3′-end of the 18 S RNA sequence to produce a 41 S molecule and a spacer-containing fragment (24 S RNA). The 41 S rRNA is cleaved forming mature 18 S rRNA and a 36 S molecule. The 36 S molecule is processed through a 32 S intermediate to the mature 28 S rRNA. This pathway is similar to that found in HeLa cells, except that in L-cells a 36 S molecule occurs in the major pathway and no 20 S precusor to 18 S RNA is found. The processing pathway and its intermediates in L-cells are analogous to those in Xenopus laevis, except for a considerable size difference in all rRNAs except 18 S rRNA. The arrangement of gene and transcribed spacer regions and of secondary structure loops, as well as the shape of the major loops were compared in L-cells, HeLa cell and Xenopus rRNA. The over-all arrangement of regions and loop patterns is very similar in the RNA from these three organisms. The shapes of loops in mature 28 S RNA are also highly conserved in evolution, but the shapes of loops in the transcribed spacer regions vary greatly. These observations suggest that the sequence complementarity that gives rise to this highly conserved secondary structure pattern may have some functional importance.