Control of gene expression by artificial introns in Saccharomyces cerevisiae.

Artificial yeast introns that show cold-sensitive splicing have been constructed. These conditional introns can be inserted into a target gene as an "intron cassette" without disrupting the coding information, allowing expression of the gene to be cold sensitive. Insertion of these intron cassettes rendered the yeast URA3 gene cold sensitive in its expression. The advantage of this intron-mediated control system is that any gene can be converted to a controllable gene by simple insertion of an intron.

Detection of somatic DNA recombination in the transgenic mouse brain.

A DNA construct containing the bacterial beta-galactosidase gene (lacZ) was used to study somatic DNA recombination in the transgenic mouse brain. Recombination-positive areas of the adult brain were stained blue with X-gal, a substrate of beta-galactosidase. Blue-colored cells appeared soon after birth, and continued to emerge in postnatal tissue. Staining was prominent in sensory as opposed to motor regions of the brain, and was present in more than 70 discrete areas of the nervous system. The possibility of DNA rearrangement is discussed with respect to the development of the central nervous system.

Mutually exclusive expression of odorant receptor transgenes.

To study the mutually exclusive expression of odorant receptor (OR) genes, we generated transgenic mice that carried the murine OR gene MOR28. Expression of the transgene and the endogenous MOR28 was distinguished by using two different markers, beta-galactosidase and green fluorescent protein (GFP), respectively. Double staining of the olfactory epithelium revealed that the two genes were rarely expressed simultaneously in individual olfactory neurons. A similar exclusion was also observed between differently tagged but identical transgenes integrated into the same locus of one particular chromosome. Although allelic inactivation has been reported for the choice between the maternal and paternal alleles, this is the first demonstration of mutually exclusive activation among non-allelic OR gene members with identical coding and regulatory sequences. Such an unusual mode of gene expression, monoallelic and mutually exclusive, has previously been shown only for the antigen-receptor genes of the immune system.

Footprint analysis of the RAG protein recombination signal sequence complex for V(D)J type recombination.

We have studied the interaction between recombination signal sequences (RSSs) and protein products of the truncated forms of recombination-activating genes (RAG) by gel mobility shift, DNase I footprinting, and methylation interference assays. Methylation interference with dimethyl sulfate demonstrated that binding was blocked by methylation in the nonamer at the second-position G residue in the bottom strand and at the sixth- and seventh-position A residues in the top strand. DNase I footprinting experiments demonstrated that RAG1 alone, or even a RAG1 homeodomain peptide, gave footprint patterns very similar to those obtained with the RAG1-RAG2 complex. In the heptamer, partial methylation interference was observed at the sixth-position A residue in the bottom strand. In DNase I footprinting, the heptamer region was weakly protected in the bottom strand by RAG1. The effects of RSS mutations on RAG binding were evaluated by DNA footprinting. Comparison of the RAG-RSS footprint data with the published Hin model confirmed the notion that sequence-specific RSS-RAG interaction takes place primarily between the Hin domain of the RAG1 protein and adjacent major and minor grooves of the nonamer DNA.

Olfactory neurons expressing closely linked and homologous odorant receptor genes tend to project their axons to neighboring glomeruli on the olfactory bulb.

We have characterized two separate odorant receptor (OR) gene clusters to examine how olfactory neurons expressing closely linked and homologous OR genes project their axons to the olfactory bulb. Murine OR genes, MOR28, MOR10, and MOR83, share 75-95% similarities in the amino acid sequences and are tightly linked on chromosome 14. In situ hybridization has demonstrated that the three genes are expressed in the same zone, at the most dorsolateral and ventromedial portions of the olfactory epithelium, and are rarely expressed simultaneously in individual neurons. Furthermore, we have found that olfactory neurons expressing MOR28, MOR10, or MOR83 project their axons to very close but distinct subsets of glomeruli on the medial and lateral sides of the olfactory bulb. Similar results have been obtained with another murine OR gene cluster for A16 and MOR18 on chromosome 2, sharing 91% similarity in the amino acid sequences. These results may indicate an intriguing possibility that olfactory neurons expressing homologous OR genes within a cluster tend to converge their axons to proximal but distinct subsets of glomeruli. These lines of study will shed light on the molecular basis of topographical projection of olfactory neurons to the olfactory bulb.

Overproduction of a gastrointestinal hormone, secretin, in Escherichia coli cells and its chemical characterization.

A cloned synthetic DNA fragment coding for 27-desamidosecretin (DAS), which differs from natural porcine secretin by the lack of the amide group at the carboxyl-terminal valine residue, was fused to the beta-galactosidase gene (lacZ) at the EcoRI site near the 3'-terminus of the gene. The fusion gene was efficiently expressed in Escherichia coli, yielding 1.15 X 10(6) fused-protein molecules per cell. After the treatment of the fused protein with cyanogen bromide, DAS was purified by a combination of ion-exchange column chromatography and reverse-phase high-performance liquid chromatography (HPLC). The structure of bacterial DAS was confirmed by tryptic mapping and amino acid composition analyses. The bacterial DAS was not amidated at its carboxyl-terminal valine residue. Nevertheless, it stimulated pancreatic secretion in rats as does authentic porcine secretin, indicating that the carboxyl-terminal amide is not essential to secretin activity.

Effect of artificially inserted intron on gene expression in Saccharomyces cerevisiae.

The intron of the yeast RP51A gene was cloned with precision using the polymerase chain reaction (PCR) amplification method, and then inserted into several different positions of the yeast URA3 gene as well as the PGK-lacZ fusion gene without introduction of additional exon sequences. Analysis of transcripts of these genes showed that an intron inserted near the transcription start site of the gene was spliced out efficiently, whereas the same intron sequences inserted 200 bp or further downstream of the start site were not, resulting in a reduced level of mRNA. These results explain why intron-containing genes in yeast usually have an intron near the 5' end.

Axonal projection of olfactory sensory neurons during the developmental and regeneration processes.

We have studied the projection of olfactory sensory neurons (OSNs), during the developmental and regeneration processes, using the transgenic mouse carrying the differently tagged odorant receptor genes, MOR28. We have found that the axon terminals of the two sets of MOR28-positive OSNs, one expressing the lacZ tag and the other expressing the green fluorescent protein gene, are dispersed and intermingled at early developmental or regeneration stages. Projection areas become more distinct and separated at later stages, however, two sets of axon fibers are not typically bundled or segregated during pathfinding. It appears that segregation of axons mainly occurs when they target at the olfactory bulb to form the glomerular structure.

The relationship between the "TATA" sequence and transcription initiation sites at the HIS4 gene of Saccharomyces cerevisiae.

Transcription of the HIS4 gene begins at a single site (I) at position -60 from the ATG that begins translation. We have made linker insertions/deletions in the 5' noncoding region to identify the elements required for the specificity of transcription initiation. Although there are four sequences that begin TATA and are near the start of transcription (-170, -132, -123, and -102) only the sequence at -123 (TATA-123) is required for transcription initiation. By inserting synthetic oligonucleotides into a mutant from which TATA-123 had been deleted, we found that just TATA or TATAA does not work but that TATAAA functions almost as well as the wild-type sequence. This hexamer does not work in the opposite orientation (TTTATA). When a synthetic TATA sequence is placed upstream from the normal site, the site of initiation also moves upstream in a roughly cometric way even when TATA-123 is present. Analysis of transcripts in strains where the distance between the TATA sequence and the wild-type site of transcription initiation (I site) has been altered shows that in yeast, unlike higher cells, transcription does not initiate at a strictly defined distance from the TATA sequence. Constructions that alter the distance between the TATA and the I site or remove the I site change the pattern of transcription initiation without affecting the level of HIS4 expression. Deletions that eliminate the I site produce heterogeneous transcripts and deletions that substantially shorten the distance between TATA-123 and the I site initiate at multiple sites downstream from the I site. Thus, both the TATA and the sequences downstream from it determine the pattern of transcription initiation.

Specific ribonucleases involved in processing of tRNA precursors of Escherichia coli. Partial purification and some properties.

Ribonucleases O and Q, the two putative nucleolytic activities which we detected previously in the crude extract from a thermosensitive ribonuclease P mutant (TS241) of Escherichia coli and which were shown to function in the processing of tRNA precursors in vitro, were partially purified from the 1000000 x g supernatant fraction of E. coli Q13. In the course of purification of these enzymes, the total RNAs synthesized in the thermosensitive mutant at the restrictive temperature were used as the substrates and the activities were identified from disappearance or alteration of specific tRNA precursor molecules in polyacrylamide gel electrophoresis. The purified ribonuclease O preparation cleaved specifically the multimeric tRNA precursors at the spacer regions. The purified ribonuclease Q preparation removed, in accordance with the definition of this enzyme, extra nucleotides from the 3'-terminal ends of monomeric tRNA precursors. Some properties of these two nucleases were investigated. In addition to these nucleases, another exonuclease (tentatively designated ribonuclease Y) and ribonuclease P, a well-characterized endonuclease, were also purified. The sequential mode of the processing of tRNA precursors, originally observed in the cleavage reactions with the crude extracts in vitro, was supported by studies with the purified enzyme preparations.

Nucleotide sequence and stability of the RNA component of RNase P from a temperature-sensitive mutant of E. coli.

The gene coding for the RNA component of RNase P was cloned from a temperature-sensitive mutant of Escherichia coli defective in RNase P activity (ts709) and its parental wild-type strain (4273), and the complete nucleotide sequences of the gene and its flanking regions were determined. The 5'- and 3'-terminal sequences of the RNA component were determined and mapped on the DNA sequence. The mutant gene has GC-to-AT substitutions at positions corresponding to 89 and 365 nucleotides downstream from the 5' terminus of the RNA sequence. Comparing to the wild-type RNA, the mutant RNA is less stable and rapidly degraded in vivo and in vitro.

Essential residues in V(D)J recombination signals.

Recombination signal sequences for V(D)J joining consist of a conserved heptamer (CACAGTG) and a nonamer (ACAAAAACC) separated by a spacer of a constant length (12 bp or 23 bp). In the present study, we have analyzed various recombination signal mutations for their effects in V(D)J joining. Using a retroviral vector, we introduced mutant substrates stably into pre-B cells, and assayed recombination using the lacZ gene as a reporter. This method allowed us to study recombination in a single copy within the context of the host cell chromosome. Because this assay did not show any detectable background, it was quite useful in the analysis of low level recombinations. In the heptamer, mutations in the first three residues severely dropped the joining rates. Among them, the first residue adjacent to the recombination site was found to be most essential. Although mutations in the heptamer reduced the joining rate to various extents, they did not lower the site-specificity of recombination. With regard to the nonamer, the presence of three consecutive A residues was necessary for efficient recombination. Furthermore, the nucleotides flanking the A-rich core needed to be other than A residues, probably marking the border of the A-stretch. This may be important when the recombinase measures the distance between the heptamer and the nonamer to satisfy the 12/23-bp spacer rule.

The PU.1 and NF-EM5 binding motifs in the Igkappa 3' enhancer are responsible for directing somatic hypermutations to the intrinsic hotspots in the transgenic Vkappa gene.

Somatic hypermutation is a key mechanism in generating Ig with higher affinities to antigen, a process known as affinity maturation. Using Igkappa transgenes, the 3' enhancer (kappaE3') has been shown to play an important role in introducing hypermutations. In order to identify the cis-acting elements that regulate hypermutagenesis, we have generated transgenic substrates containing mutations/deletions in the kappaE3' region. Here, we report that base substitutions in the kappaE3', either in the PU.1 or in the NF-EM5 binding motif, not only reduce the mutation rate but also disrupt the directed mutagenesis in the intrinsic hotspots of the Igkappa transgene.

The PU.1 binding site is a cis-element that regulates pro-B/pre-B specificity of Vkappa-Jkappa joining.

We have previously shown that the PU.1 binding motif (GAG GAA) in the 3'-enhancer region in the Ig kappa gene is responsible for the negative regulation of tissue (B/T)-specific Vkappa-Jkappa joining. Here we report that the PU.1 binding site also regulates the stage (pro-B/pre-B) specificity of Vkappa-Jkappa joining. In the substrate with base substitutions in the PU.1 binding motif, recombination took place in both pro-B (B220dull/CD43+) and pre-B (B220dull/CD43-) cells. In the transcriptional regulation, the PU.1 motif acts in a positive manner cooperatively with the nuclear factor-EM5 (or PIP) motif (GAAAAC), which is located 2 bp downstream from the PU.1 motif. Interestingly, base substitutions in the nuclear factor EM5 (PIP) motif did not affect the pro-B/pre-B specificity of Vkappa-Jkappa joining. Thus, the PU.1 motif regulates both temporal and tissue-specific rearrangements, while nuclear factor-EM5 is not involved in the regulation of Ig kappa recombination.

The DNA-bending protein, HMG1, is required for correct cleavage of 23 bp recombination signal sequences by recombination activating gene proteins in vitro.

DNA-bending proteins are known to facilitate the in vitro V(D)J joining of antigen receptor genes. Here we report that the high-mobility group protein, HMG1, is necessary for the correct nicking of the 23 bp recombination signal sequence (23-RSS) by the recombination [corrected] activating gene (RAG) proteins, RAG1 and RAG2. Without HMG1, the mouse Jkappa1 23-RSS was recognized as if it were the 12-RSS and nicked at a site 12 + 7 nucleotides away from the 9mer signal, even though no 7mer-like sequence was evident at the cryptic nicking site. When increased amounts of HMG1 were added, the 23-RSS substrate was nicked correctly at a site 23 + 7 nucleotides from the 9mer, and nicking at the cryptic site disappeared. Unlike the 23-RSS, the 12-RSS did not require HMG1 for correct nicking, although HMG1 was found to increase the interaction between RSS and RAG proteins. Modification-interference assays demonstrated that HMG1 caused changes in the interaction between the 23-RSS and RAG proteins specifically at the 7mer and the cryptic nicking site.