Control of Human PLP1 Expression Through Transcriptional Regulatory Elements and Alternatively Spliced Exons in Intron 1

Although the myelin proteolipid protein gene (PLP1) encodes the most abundant protein in central nervous system (CNS) myelin, not much is known about the mechanisms that govern expression of the human gene (hPLP1). Much more is known about the processes that regulate Plp1 gene expression in rodents. From studies with Plp1-lacZ transgenic mice, it was determined that the first intron of mouse Plp1 (mPlp1) is required to attain high levels of expression in brain, concurrent with the active myelination period. Other studies have suggested that within mPlp1 intron 1 (>8 kb) lie several regions with enhancer-like activity. To test whether these sequences (and possibly others) in hPLP1 intron 1 are functional, deletion-transfection analysis was performed with hPLP1-lacZ constructs that contain various portions of the intron, or lack it altogether. Results presented here demonstrate the importance of hPLP1 intron 1 in achieving maximal levels of expression in the immortalized oligodendroglial cell line, Oli-neu. Deletion analysis indicates that the intron contains multiple positive regulatory elements which are active in Oli-neu cells. Some of these elements appear to be functionally conserved between human and mouse, while others are not. Furthermore, our studies demonstrate that multiple splice variants can be formed due to inclusion of extra (supplementary) exons from what is classically thought of as hPLP1 intron 1. Thus, splicing of these novel exons (which are not recognized as such in mPlp1 due to lack of conserved splice sites) must utilize factors common to both human and mouse since Oli-neu cells are of mouse origin.     

Targeted deletion of the antisilencer/enhancer (ASE) element from intron 1 of the myelin proteolipid protein gene (Plp1) in mouse reveals that the element is dispensable for Plp1 expression in brain during development and remyelination

Myelin proteolipid protein gene (Plp1) expression is temporally regulated in brain, which peaks during the active myelination period of CNS development. Previous studies with Plp1‐lacZ transgenic mice demonstrated that (mouse) Plp1 intron 1 DNA is required for high levels of expression in oligodendrocytes. Deletion‐transfection analysis revealed the intron contains a single positive regulatory element operative in the N20.1 oligodendroglial cell line, which was named ASE (a ntis ilencer/e nhancer) based on its functional properties in these cells. To investigate the role of the ASE in vivo, the element was deleted from the native gene in mouse using a Cre/lox strategy. Although removal of the ASE from Plp1‐lacZ constructs profoundly decreased expression in transfected oligodendroglial cell lines (N20.1 and Oli‐neu), the element was dispensable to achieve normal levels of Plp1 gene expression in mouse during development (except perhaps at postnatal day 15) and throughout the remyelination period following cuprizone‐induced (acute) demyelination. Thus, it is possible that the ASE is non‐functional in vivo, or that loss of the ASE from the native gene in mouse can be compensated for by the presence of other regulatory elements within the Plp1 gene.     

Gtl2 lacZ,an insertional mutation on mouse Chromosome 12 with parental origin-dependent phenotype

We have produced a transgenic mouse line, Gtl2 ^lacZ (Gene trap locus 2), that carries an insertional mutation with a dominant modified pattern of inheritance:heterozygous Gtl2 ^lacZ mice that inherited the transgene from the father show a proportionate dwarfism phenotype, whereas the penetrance and expressivity of the phenotype is strongly reduced in Gtl2 ^lacZ mice that inherited the transgene from the mother. On a mixed genetic background this pattern of inheritance was reversible upon transmission of the transgene through the germ line of the opposite sex. On a predominantly 129/Sv genetic background, however, transgene passage through the female germ line modified the transgene effect, such that the penetrance of the mutation was drastically reduced and the phenotype was no longer obvious after subsequent male germ line transmission. Expression of the transgene, however, was neither affected by genetic background nor by parental legacy. Gtl2 ^lacZ maps to mouse Chromosome 12 in a region that displays imprinting effects associated with maternal and paternal disomy. Our results suggest that the transgene insertion in Gtl2 ^lacZ mice affects an endogenous gene(s) required for fetal and postnatal growth and that this gene(s) is predominantly paternally expressed. Received: 30 May 1995 / Accepted: 7 August 1995     

A myelin proteolipid protein-LacZ fusion protein is developmentally regulated and targeted to the myelin membrane in transgenic mice

Transgenic mice were generated with a fusion gene carrying a portion of the murine myelin proteolipid protein (PLP) gene, including the first intron, fused to the E. coli LacZ gene. Three transgenic lines were derived and all lines expressed the transgene in central nervous system white matter as measured by a histochemical assay for the detection of beta-galactosidase activity. PLP-LacZ transgene expression was regulated in both a spatial and temporal manner, consistent with endogenous PLP expression. Moreover, the transgene was expressed specifically in oligodendrocytes from primary mixed glial cultures prepared from transgenic mouse brains and appeared to be developmentally regulated in vitro as well. Transgene expression occurred in embryos, presumably in pre- or nonmyelinating cells, rather extensively throughout the peripheral nervous system and within very discrete regions of the central nervous system. Surprisingly, beta- galactosidase activity was localized predominantly in the myelin in these transgenic animals, suggesting that the NH2-terminal 13 amino acids of PLP, which were present in the PLP-LacZ gene product, were sufficient to target the protein to the myelin membrane. Thus, the first half of the PLP gene contains sequences sufficient to direct both spatial and temporal gene regulation and to encode amino acids important in targeting the protein to the myelin membrane.     

Different cis-Regulatory DNA Elements Mediate Developmental Stage- and Tissue-specific Expression of the Human COL2A1 Gene in Transgenic Mice

Expression of the type II collagen gene (human COL2A1, mouse Col2a1) heralds the differentiation of chondrocytes. It is also expressed in progenitor cells of some nonchondrogenic tissues during embryogenesis. DNA sequences in the 5′ flanking region and intron 1 are known to control tissue-specific expression in vitro, but the regulation of COL2A1 expression in vivo is not clearly understood. We have tested the regulatory activity of DNA sequences from COL2A1 on the expression of a lacZ reporter gene in transgenic mice. We have found that type II collagen characteristic expression of the transgene requires the enhancer activity of a 309-bp fragment (+2,388 to +2,696) in intron 1 in conjunction with 6.1-kb 5′ sequences. Different regulatory elements were found in the 1.6-kb region (+701 to +2,387) of intron 1 which only needs 90-bp 5′ sequences for tissue-specific expression in different components of the developing cartilaginous skeleton. Distinct positive and negative regulatory elements act together to control tissue-specific transgene expression in the developing midbrain neuroepithelium. Positive elements affecting expression in the midbrain were found in the region from −90 to −1,500 and from +701 to +2,387, whereas negatively acting elements were detected in the regions from −1,500 to −6,100 and +2,388 to +2,855.     

Mouse arylamine N-acetyltransferase 2 (Nat2) expression during embryogenesis: a potential marker for the developing neuroendocrine system

Arylamine N-acetyltransferase (NAT) genes in humans and in rodents encode polymorphic drug metabolizing enzymes. Human NAT1 (and the murine equivalent mouse Nat2) is found early in embryonic development and is likely to have an endogenous role. We report the detailed expression of the murine gene (Nat2) and encoded protein in mouse embryos, using a transgenic mouse model bearing a lacZ transgene inserted into the coding region of mouse Nat2. In mouse embryos, the transgene was expressed in sensory epithelia, epithelial placodes giving rise to visceral sensory neurons, the developing pituitary gland, sympathetic chain and urogenital ridge. In Nat2^+/+ mice, the presence and activity of Nat2 protein was detected in these tissues and their adult counterparts. Altered expression of the human orthologue in breast tumours, in which there is endocrine signalling, suggests that human NAT1 should be considered as a potential biomarker for neuroendocrine tissues and tumours.     

The specificity of the myelin basic protein gene promoter studied in transgenic mice.

The myelin basic proteins (MBPs) are a family of polypeptides that are predominantly expressed in the nervous system where they play a major role in myelination. We have generated four lines of transgenic mice carrying a transgene in which 1.34 kb of the 5'-flanking sequence of the mouse MBP gene was fused upstream of the coding region of the Escherichia coli lac Z gene in order to investigate developmental and tissue-specific expression of the MBP gene. Expression of both the lacZ transgene and the endogenous MBP gene followed a common developmental pattern in mouse brain. Transgene expression was detected in primary oligodendrocytes, but not in type 2 astrocytes. In addition, the lacZ gene product was expressed in epithelial cells of certain nonneural tissues, namely kidney, epididymis, ureter, and seminal vesicles. The ectopic expression of the transgene was associated with the development of DNase I hypersensitive sites at the site of insertion which was found to be within the intron 1 region of the endogenous MBP gene. The results reported here strongly suggest that the 1.34-kb 5'-flanking region of the MBP gene contains cis-regulatory elements that confer developmental regulation of the MBP gene, although this region appears to lack elements that restrict its expression to the nervous system.     

Parental imprinting of an IGF-2 transgene

As a consequence of parental imprinting in mice, the paternal allele encoding insulin-like growth factor-II (IGF-II) is expressed, whereas the maternal allele is silent in most tissues. To examine whether cis-acting sequences involved in imprinting are located in the vicinity of the lgf-2 gene, we have constructed mouse transgenic lines and studied the expression of a 30 kb rat lgf-2 transgene, in which the coding region has been replaced with the lacZ reporter sequence. Chromatin position effects and/or absence of long-range regulatory elements seem to have affected tissue-specific expression in the transgenic mice. However, in one of six expressing lines, staining of embryos for β-galactosidase activity was detected in a minor subset of tissues normally transcribing the endogenous homolog, but only when the transgene was transmitted paternally. This transgene was integrated into mouse chromosome 19, which is apparently free of imprinted loci. Although the possibility that the lgf-2 transgene was inserted into an as yet unidentified imprinted iocus is discussed, a more likely interpretation of our results is that the transgene carries at least a portion of its own imprinting signal, because it consists of the genomic sequences of a locus already known to be imprinted and maintains the correct imprinting mode. © 1993 Wiley-Liss, Inc.     

Functional characterization of a cis-acting DNA antisilencer region that modulates myelin proteolipid protein gene expression

Regulation of myelin proteolipid protein (PLP:) gene expression is tightly controlled, both spatially and temporally. Previously, we have shown with transgenic mice that a PLP:-lacZ fusion gene (which includes the entire sequence for PLP: intron 1 DNA) is regulated in a similar manner to endogenous PLP: gene expression. Furthermore, by deletion-transfection analyses using assorted PLP:-lacZ constructs with partial deletion of PLP: intron 1 sequences, we have shown that the first intron possesses an antisilencer region that is capable of over-coming repression mediated by two distinct regions located elsewhere within intron 1 DNA. Here, we report the ability of various fragments encompassing the antisilencer region to restore beta-galactosidase activity when inserted into PLP:-lacZ constructs, which originally exhibited low levels of beta-galactosidase activity. Additional constructs were generated to test the effects of these antisilencer-containing fragments in constructs that are missing either one or both of the negative regulatory regions that are overridden during antisilencing. Transfection analyses, in conjunction with protein-DNA binding assays, suggest that several nuclear factors are necessary for derepression of PLP: gene activity in an oligodendroglial cell line. Moreover, either the "core" or complete antisilencing region can act in an additive or synergistic fashion when multiple copies are inserted into the Plp-lacZ constructs.     

Plasmids expressing the Herpes simplex virus thymidine kinase gene in mammalian and bacterial cells

Summary Two plasmids containing either the complete thymidine kinase gene of Herpes simplex virus type I (pSK2) or the gene without the remote control sequence (pSK1) just behind the lac promoter and the first codons of the lacZ gene were constructed. Both plasmids efficiently transform mouse Ltk^- cells as well as E. coli tk^- cells to the Tk⁺ phenotype and are well suited for plasmid rescue from transformed mouse cells by direct functional selection for tk expression using a tk ^- mutant of E. coli C600.     

Loss of imprinting at the Dlk1-Gtl2 locus caused by insertional mutagenesis in the Gtl2 5' region

Background

The Dlk1 and Gtl2 genes define a region of mouse chromosome 12 that is subject to genomic imprinting, the parental allele-specific expression of a gene. Although imprinted genes play important roles in growth and development, the mechanisms by which imprinting is established and maintained are poorly understood. Differentially methylated regions (DMRs), which carry methylation on only one parental allele, are involved in imprinting control at many loci. The Dlk1-Gtl2 region contains three known DMRs, the Dlk1 DMR in the 3' region of Dlk1, the intergenic DMR 15 kb upstream of Gtl2, and the Gtl2 DMR at the Gtl2 promoter. Three mouse models are analyzed here that provide new information about the regulation of Dlk1-Gtl2 imprinting.

Results

A previously existing insertional mutation (Gtl2lacZ), and a targeted deletion in which the Gtl2 upstream region was replaced by a Neo cassette (Gtl2Δ5'Neo), display partial lethality and dwarfism upon paternal inheritance. Molecular characterization shows that both mutations cause loss of imprinting and changes in expression of the Dlk1, Gtl2 and Meg8/Rian genes. Dlk1 levels are decreased upon paternal inheritance of either mutation, suggesting Dlk1 may be causative for the lethality and dwarfism. Loss of imprinting on the paternal chromosome in both Gtl2lacZ and Gtl2Δ5'Neo mice is accompanied by the loss of paternal-specific Gtl2 DMR methylation, while maternal loss of imprinting suggests a previously unknown regulatory role for the maternal Gtl2 DMR. Unexpectedly, when the Neo gene is excised, Gtl2Δ5' animals are of normal size, imprinting is unchanged and the Gtl2 DMR is properly methylated. The exogenous DNA sequences integrated upstream of Gtl2 are therefore responsible for the growth and imprinting effects.

Conclusion

These data provide further evidence for the coregulation of the imprinted Dlk1 and Gtl2 genes, and support a role for Dlk1 as an important neonatal growth factor. The ability of the Gtl2lacZ and Gtl2Δ5'Neo mutations to cause long-range changes in imprinting and gene expression suggest that regional imprinting regulatory elements may lie in proximity to the integration site.     

Recombinant Rp1 genes confer necrotic or nonspecific resistance phenotypes

Genes at the Rp1 rust resistance locus of maize confer race-specific resistance to the common rust fungus Puccinia sorghi. Three variant genes with nonspecific effects (HRp1 -Kr1N, -D*21 and -MD*19) were found to be generated by intragenic crossing over within the LRR region. The LRR region of most NBS-LRR encoding genes is quite variable and codes for one of the regions in resistance gene proteins that controls specificity. Sequence comparisons demonstrated that the Rp1-Kr1N recombinant gene was identical to the N-terminus of the rp1-kp2 gene and C-terminus of another gene from its HRp1-K grandparent. The Rp1-D*21 recombinant gene consists of the N-terminus of the rp1-dp2 gene and C-terminus of the Rp1-D gene from the parental haplotype. Similarly, a recombinant gene from the Rp1-MD*19 haplotype has the N-terminus of an rp1 gene from the HRp1-M parent and C-terminus of the rp1-D19 gene from the HRp1-D parent. The recombinant Rp1 -Kr1N, -D*21 and -MD*19 genes activated defense responses in the absence of their AVR proteins triggering HR (hypersensitive response) in the absence of the pathogen. The results indicate that the frequent intragenic recombination events that occur in the Rp1 gene cluster not only recombine the genes into novel haplotypes, but also create genes with nonspecific effects. Some of these may contribute to nonspecific quantitative resistance but others have severe consequences for the fitness of the plant.     

A Combination of PLP and DM20 Transgenes Promotes Partial Myelination in the Jimpy Mouse

Abstract: Mutations in the myelin proteolipid protein (PLP) gene, such as that found in the jimpy mouse, result in an abnormal structure of the myelin, severe dysmyelination, and a reduction in the number of mature oligodendrocytes. To examine the functions of the two alternatively spliced isoforms of proteolipid protein, transgenic mice were generated that express either PLP or DM20 cDNAs placed under control of the PLP upstream regulatory region. The transgenes were bred into jimpy mice, and the effect of the transgenes on the dysmyelinating phenotype was analyzed. Neither the PLP transgene nor the DM20 transgene alone had an effect on myelination in the jimpy mice. Combining the two transgenes substantially increased the number of myelinated axons, suggesting that the two alternatively spliced products of the PLP locus perform distinct functions in oligodendrocytes. The enhanced myelination was not sufficient, however, for completely correcting the dysmyelinating phenotype of the jimpy mice, nor was it accompanied by the restoration of normal levels of myelin gene expression. The inability to rescue the jimpy phenotype is most likely attributable to a dominant negative action of the abnormal proteolipid proteins present in jimpy mice. These results demonstrate the complexity of proteolipid protein function in myelination.     

Inactivation of the ecdysteroid UDP-glucosyltransferase (egt) gene of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) improves its virulence towards its insect host

Some baculovirus have been genetically modified for the inactivation of their ecdysteroid glucosyltransferase (egt) gene, and these viruses were shown to kill infected larvae more rapidly when compared to wild-type virus infections. We have previously identified, cloned, and sequenced the egt gene of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV). Here we present data regarding the construction of an egt minus (egt−) AgMNPV and its virulence towards its insect host. We have inserted an hsp70-lacZ (3.7 kb) gene cassette into the egt gene open reading frame (ORF) and purified a recombinant AgMNPV (vAgEGTΔ-lacZ). Bioassays with third-instar A. gemmatalis larvae showed that viral occlusion body (OB) production were consistently lower from infections with vAgEGTΔ-lacZ compared to the wild-type virus. A mean of 20.4×10⁸ OBs/g/larva and 40.7×10⁸ OBs/g/larva was produced from vAgEGTΔ-lacZ and AgMNPV infections, respectively. The mean lethal concentration which killed 50% of insects in a treatment group (LC₅₀) for the 10th day after virus treatment (DAT) was 3.9-fold higher for the wild-type virus compared to vAgEGTΔ-lacZ. The recombinant virus killed A. gemmatalis larvae significantly faster (ca. 1–2.8 days), than the wild-type AgMNPV. Therefore, the vAgEGTΔ-lacZ was more efficacious for the control of A. gemmatalis larvae (in bioassays) compared to wild-type AgMNPV.