Jürgen Brosius

Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli

A strong promoter has been cloned on a series of plasmid vectors that facilitate the expression of cloned genes. This promoter, named tac [first described by DeBoer et al. (in Rodriguez, R.L. and Chamberlin, M.J. (Eds.),Promoters, Structure and Function. Praeger, New York, 1982, pp. pp. 462-481)] contains the -10 region of the lacUV5 promoter and the -35 region of the trp promoter. Our vectors contain various cloning sites followed by transcription termination signals. In addition, we describe plasmids that facilitate the conversion of the lac promoter to the stronger tac promoter. Thus, preexisting gene fusions using the lac or the lacUV5 promoter can be readily converted to tac promoter gene fusions without changing the ribosome-binding site (RBS). The tac promoter is repressed in lacIQ strains and can be induced by isopropylthio-beta-D-galactoside (IPTG). Studies of expression of the cI repressor of bacteriophage lambda show that the tac promoter is at least five times more efficient than the lacUV5 promoter. Under optimal conditions lambda repressor constitutes up to 30% of the total cellular protein.

‘ATG vectors’ for regulated high-level expression of cloned genes in Escherichia coli

A plasmid cloning vector system has been constructed that allows for the production of large quantities of foreign proteins or fragments thereof, in an unfused state. These vectors provide strong regulated trp-lac fusion promoters and the lacZ ribosome-binding site (RBS) followed by an ATG translation initiation codon at an appropriate distance from the RBS. The ATG codon is located within a unique NcoI restriction site (CCATGG). Digestion with NcoI exposes the ATG for fusion. Gene fragments lacking a prokaryotic RBS and/or ATG start codons can be inserted in several ways. Expression experiments using a truncated cI gene of bacteriophage lambda or a large portion of the coding region of the Herpes simplex virus type 1 glycoprotein D gene have been performed. The results of these studies show that the vectors are useful for the high-level expression of prokaryotic and eukaryotic genes in Escherichia coli.

RNAs from all categories generate retrosequences that may be exapted as novel genes or regulatory elements.

While the significance of middle repetitive elements had been neglected for a long time, there are again tendencies to ascribe most members of a given middle repetitive sequence family a functional role--as if the discussion of SINE (short interspersed repetitive elements) function only can occupy extreme positions. In this article, I argue that differences between the various classes of retrosequences concern mainly their copy numbers. Consequently, the function of SINEs should be viewed as pragmatic such as, for example, mRNA-derived retrosequences, without underestimating the impact of retroposition for generation of novel protein coding genes or parts thereof (exon shuffling by retroposition) and in particular of SINEs (and retroelements) in modulating genes and their expression. Rapid genomic change by accumulating retrosequences may even facilitate speciation [McDonald, J.F., 1995. Transposable elements: possible catalysts of organismic evolution. Trends Ecol. Evol. 10, 123-126.] In addition to providing mobile regulatory elements, small RNA-derived retrosequences including SINEs can, in analogy to mRNA-derived retrosequences, also give rise to novel small RNA genes. Perhaps not representative for all SINE/master gene relationships, we gained significant knowledge by studying the small neuronal non-messenger RNAs, namely BC1 RNA in rodents and BC200 RNA in primates. BC1 is the first identified master gene generating a subclass of ID repetitive elements, and BC200 is the only known Alu element (monomeric) that was exapted as a novel small RNA encoding gene.

Plasmid vectors for the selection of promoters

Vectors were constructed which contain promoterless genes for chloramphenicol (cam) or tetracycline (tet) resistance, as promoter-probe plasmids. Escherichia coli cells harboring these plasmids are sensitive to cam or tet but resistant to ampicillin. In plasmids pKK231 -1 and pKK232 -8 the gene for cam acetyltransferase (CAT) and in pKK175 -6 the gene for tet resistance are flanked by efficient transcription terminators, preventing transcription from other pBR322 promoters into the antibiotic resistance region. In one of the vectors, pKK232 -8, translational stop codons were introduced in all three reading frames upstream from the initiation codon of the cat gene. If a DNA fragment containing a promoter is inserted into one of the cloning sites upstream from the antibiotic genes, cells carrying such plasmids acquire resistance to cam or tet. Using these vectors two restriction fragments that contain promoters were identified. One of these fragments contains sequences upstream from an unidentified gene ( ORFII ) located distal to the rrnB rRNA operon of E. coli.

RNomics: an experimental approach that identifies 201 candidates for novel, small, non‐messenger RNAs in mouse

In mouse brain cDNA libraries generated from small RNA molecules we have identified a total of 201 different expressed RNA sequences potentially encoding novel small non‐messenger RNA species (snmRNAs). Based on sequence and structural motifs, 113 of these RNAs can be assigned to the C/D box or H/ACA box subclass of small nucleolar RNAs (snoRNAs), known as guide RNAs for rRNA. While 30 RNAs represent mouse homologues of previously identified human C/D or H/ACA snoRNAs, 83 correspond to entirely novel snoRNAs. Among these, for the first time, we identified four C/D box snoRNAs and four H/ACA box snoRNAs predicted to direct modifications within U2, U4 or U6 small nuclear RNAs (snRNAs). Furthermore, 25 snoRNAs from either class lacked antisense elements for rRNAs or snRNAs. Therefore, additional snoRNA targets have to be considered. Surprisingly, six C/D box snoRNAs and one H/ACA box snoRNA were expressed exclusively in brain. Of the 88 RNAs not belonging to either snoRNA subclass, at least 26 are probably derived from truncated heterogeneous nuclear RNAs (hnRNAs) or mRNAs. Short interspersed repetitive elements (SINEs) are located on five RNA sequences and may represent rare examples of transcribed SINEs. The remaining RNA species could not as yet be assigned either to any snmRNA class or to a part of a larger hnRNA/mRNA. It is likely that at least some of the latter will represent novel, unclassified snmRNAs.

Identification of 86 candidates for small non-messenger RNAs from the archaeon Archaeoglobus fulgidus

In a specialized cDNA library from the archaeon Archaeoglobus fulgidus we have identified a total of 86 different expressed RNA sequences potentially encoding previously uncharacterized small non-messenger RNA (snmRNA) species. Ten of these RNAs resemble eukaryotic small nucleolar RNAs, which guide rRNA 2′-O-methylations (C/D-box type) and pseudouridylations (H/ACA-box type). Thereby, we identified four candidates for H/ACA small RNAs in an archaeal species that are predicted to guide a total of six rRNA pseudouridylations. Furthermore, we have verified the presence of the six predicted pseudouridines experimentally. We demonstrate that 22 snmRNAs are transcribed from a family of short tandem repeats conserved in most archaeal genomes and shown previously to be potentially involved in replicon partitioning. In addition, four snmRNAs derived from the rRNA operon of A. fulgidus were identified and shown to be generated by a splicing/processing pathway of pre-rRNAs. The remaining 50 RNAs could not be assigned to a known class of snmRNAs because of the lack of known structure and/or sequence motifs. Regarding their location on the genome, only nine were located in intergenic regions, whereas 33 were complementary to an ORF, five were overlapping an ORF, and three were derived from the sense orientation within an ORF. Our study further supports the importance of snmRNAs in all three domains of life.

Retroposed elements as archives for the evolutionary history of placental mammals.

Reconstruction of the placental mammalian (eutherian) evolutionary tree has undergone diverse revisions, and numerous aspects remain hotly debated. Initial hierarchical divisions based on morphology contained many misgroupings due to features that evolved independently by similar selection processes. Molecular analyses corrected many of these misgroupings and the superordinal hierarchy of placental mammals was recently assembled into four clades. However, long or rapid evolutionary periods, as well as directional mutation pressure, can produce molecular homoplasies, similar characteristics lacking common ancestors. Retroposed elements, by contrast, integrate randomly into genomes with negligible probabilities of the same element integrating independently into orthologous positions in different species. Thus, presence/absence analyses of these elements are a superior strategy for molecular systematics. By computationally scanning more than 160,000 chromosomal loci and judiciously selecting from only phylogenetically informative retroposons for experimental high-throughput PCR applications, we recovered 28 clear, independent monophyly markers that conclusively verify the earliest divergences in placental mammalian evolution. Using tests that take into account ancestral polymorphisms, multiple long interspersed elements and long terminal repeat element insertions provide highly significant evidence for the monophyletic clades Boreotheria (synonymous with Boreoeutheria), Supraprimates (synonymous with Euarchontoglires), and Laurasiatheria. More importantly, two retropositions provide new support for a prior scenario of early mammalian evolution that places the basal placental divergence between Xenarthra and Epitheria, the latter comprising all remaining placentals. Due to its virtually homoplasy-free nature, the analysis of retroposon presence/absence patterns avoids the pitfalls of other molecular methodologies and provides a rapid, unequivocal means for revealing the evolutionary history of organisms.

Structural relationship of human interferon alpha genes and pseudogenes

We have isolated and characterized DNA segments containing IFN-alpha-related sequences from human lambda and cosmid clone banks. We describe six linkage groups comprising 18 distinct IFN-alpha-related loci, and report the nucleotide sequences of nine chromosomal IFN-alpha-genes with intact reading frames, as well as of five pseudogenes. Taking into account as yet unsequenced genes as well as clones described by others, there are now seven linkage groups and 23 loci, of which 15 correspond to potentially functional genes and six to non-functional genes; two loci remain unsequenced. Eighteen additional sequences are likely to be allelic to the above. The finding that at least two IFN-alpha genes appear to be natural hybrids of other IFN-alpha genes, and that two distinct IFN-alpha loci have completely identical coding sequences, although their flanking regions are different, is evidence for information exchange between the individual genes.

Toxicity of an overproduced foreign gene product in Escherichia coli and its use in plasmid vectors for the selection of transcription terminators

A rat insulin gene which was fused to Escherichia coli signals for the initiation of translation could not be retained when expressed from the strong rrnB ribosomal RNA promoters or an induced trp/lac (= tac) hybrid promoter. When the latter promoter was repressed by transformation into a lac-repressor-overproducing strain, the insulin gene fragment could be retained. Upon induction of the promoter with isopropyl-beta-D-galactosidase the growth rate of the cells was reduced, and in most cases the cells subsequently lysed. Deletion of the translational initiation signals, changing the reading frame, or insertion of an efficient transcription terminator between the promoter and the rat insulin gene each permitted retention of the fragment. The first two observations indicate that overproduction of the specific polypeptide, and not of the RNA, is detrimental to the cell. The third finding has been exploited for the testing and selection of transcription terminators. The rpoC terminator, which is located distal to the rplJL /rpoBC operon, has been shown to terminate transcripts from the rrnB promoters. It was also shown that the putative rrnB terminators, T1 and T2, each function separately in vivo.

Expression of neural BC200 RNA in human tumours

BC200 RNA is a 200‐nucleotide‐long non‐messenger RNA that is selectively expressed in the primate nervous system, where it has been identified in somatodendritic domains of a subset of neurons. BC200 RNA is not normally expressed in non‐neuronal somatic cells; it has been shown, however, to be expressed in germ cells and in cultured immortal cell lines of various non‐neural origins. In order to investigate whether the neuron‐specific expression of BC200 RNA is also deregulated during tumourigenesis in non‐neural human tissues, 80 different tumour specimens, representing 19 different tumour types, were screened for the presence of the RNA. BC200 RNA was expressed in carcinomas of the breast, cervix, oesophagus, lung, ovary, parotid, and tongue, but not in corresponding normal tissues. BC200 RNA was not detectable in bladder, colon, kidney, or liver carcinoma tissues examined in this study. These results demonstrate that BC200 expression is deregulated under certain neoplastic conditions. The expression of BC200 RNA in non‐neural tumours may indicate a functional interrelationship with induction and/or progression of such tumours.