The Large Isoform of Myelin-Associated Glycoprotein Is Scarcely Expressed in the Quaking Mouse Brain

Two polypeptide isoforms of myelin-associated glycoprotein (MAG) with molecular masses of 72 and 67 kDa are produced by alternative splicing of the exon 12 portion. Our previous work has demonstrated that in the quaking mouse brain this alternative splicing is lacking and that the mRNA coding the large MAG isoform (L-MAG) is scarcely expressed, whereas that of small MAG isoform (S-MAG) is overexpressed. In the present study, we prepared antisera specific to the S-MAG and L-MAG amino acid residues, respectively. Immunoblots showed that the L-MAG band was scarcely detectable in the quaking mouse brain, whereas the S-MAG band had an apparently higher molecular mass than in the normal control. Our immunohistochemical study also showed that L-MAG was scarcely stained in the quaking mouse brain. These results seemed to reflect a reduction in content of L-MAG mRNA and abnormal glycosylation in the quaking mouse brain.     

cDNA cloning of mouse myelin-associated glycoprotein: a novel alternative splicing pattern

The structures of three forms of mouse myelin-associated glycoprotein mRNAs were determined from full-length cDNA clones. Two forms of mRNAs have been reported to be different by alternate inclusion of exon 2 and 12 in rat brain. One of the three forms of clones obtained here appeared to be a novel mRNA which lacked both the exon 2 and 12 portions, although others were identical splicing patterns to those of rat. Northern blot analysis using specific probes to mRNAs with or without the exon 2 portion in normal and quaking mouse confirmed that the splicing of exon 2 and 12 occurred independently.     

An intron element modulating 5' splice site selection in the hnRNP A1 pre-mRNA interacts with hnRNP A1.

The hnRNP A1 pre-mRNA is alternatively spliced to yield the A1 and A1b mRNAs, which encode proteins differing in their ability to modulate 5' splice site selection. Sequencing a genomic portion of the murine A1 gene revealed that the intron separating exon 7 and the alternative exon 7B is highly conserved between mouse and human. In vitro splicing assays indicate that a conserved element (CE1) from the central portion of the intron shifts selection toward the distal donor site when positioned in between the 5' splice sites of exon 7 and 7B. In vivo, the CE1 element promotes exon 7B skipping. A 17-nucleotide sequence within CE1 (CE1a) is sufficient to activate the distal 5' splice site. RNase T1 protection/immunoprecipitation assays indicate that hnRNP A1 binds to CE1a, which contains the sequence UAGAGU, a close match to the reported optimal A1 binding site, UAGGGU. Replacing CE1a by different oligonucleotides carrying the sequence UAGAGU or UAGGGU maintains the preference for the distal 5' splice site. In contrast, mutations in the AUGAGU sequence activate the proximal 5' splice site. In support of a direct role of the A1-CE1 interaction in 5'-splice-site selection, we observed that the amplitude of the shift correlates with the efficiency of A1 binding. Whereas addition of SR proteins abrogates the effect of CE1, the presence of CE1 does not modify U1 snRNP binding to competing 5' splice sites, as judged by oligonucleotide-targeted RNase H protection assays. Our results suggest that hnRNP A1 modulates splice site selection on its own pre-mRNA without changing the binding of U1 snRNP to competing 5' splice sites.     

Mouse connexin 45: genomic cloning and exon usage

Connexin 45 is a gap junction protein that is prominent in early embryos and is widely expressed in many mature cell types. To elucidate its gene structure, expression, and regulation, we isolated mouse Cx45 genomic clones. Alignment of the genomic DNA and cDNA sequences revealed the presence of three exons and two introns. The first two exons contained only 5' untranslated sequences, while exon 3 contained the remaining 5' UTR, the entire coding region, and the 3' UTR. An RT-PCR with exon-specific primers was utilized to examine exon usage in F9 mouse embryonal carcinoma cells and adult mouse tissues. In all samples, PCR products amplified using exon 2/exon 3 or exon 3/exon 3 primer pairs were much more abundant than products produced using exon 1/exon 2 or exon 1/exon 3 primer pairs, suggesting that Cx45 mRNAs containing exon 1 were relatively rare compared with mRNAs containing the other exons. Rapid amplification of cDNA ends (5'-RACE) was performed using antisense primers from within exon 3 and template RNA prepared from F9 cells or from adult mouse kidney. We obtained multiple RACE products from both templates, including products that contained all three exons and were spliced identically to the cDNA. However, clones were also isolated (from kidney) that began within the region previously identified as intron 1 and continued upstream with a sequence identical to the cDNA, including splicing to exon 3. These results show that mouse Cx45 has a gene structure that differs from that of previously studied connexins and allows the production of heterogeneous Cx45 mRNAs with differing 5' UTRs. These differences might contribute to regulation of Cx45 protein levels by modulating mRNA stability or translational efficiency.     

Nucleotide sequence of complementary DNA encoding for quaking protein of cow, horse and pig.

Complementary DNA (cDNA) for bovine quaking gene (Bqk), equine quaking gene (Eqk) and porcine quaking gene (Pqk), which are homologous to mouse quaking gene (qkI), were isolated, and their nucleotide sequences were determined. cDNA sequences of Bqk, Eqk and Pqk showed very high homology to that of qkI at nucleotide level; 94.2, 95.7 and 95.6%, respectively. Deduced amino acid sequences for Bqk, Eqk and Pqk perfectly matched to that of qkI. These findings suggest that the quaking gene family is highly conserved during mammalian evolution, and that Bqk, Eqk and Pqk are likely to have important biological functions also in cow, horse and pig.     

Studies of brain myelin in the "quaking mouse"

Myelin was isolated from the brains of "quaking" and littermate control animals and its composition was determined. The brains of quaking animals contained approximately one-fourth as much myelin as the control animals. There were qualitative as well as quantitative differences between the myelin from the two groups. By continuous cesium chloride gradient flotation it was shown that the myelin from the quaking animals consisted solely of a band corresponding to the heavier and smaller of the two bands found in normal controls. Cholesterol and glycolipids were lower and phospholipids (mainly phosphatidylcholine) and protein were higher in quaking animals than in controls. Also, phosphatidal-ethanolamine was decreased, and several consistent differences in the fatty acids (both unsubstituted and hydroxy) and aldehydes of the component lipids were found. In general there were smaller amounts of monounsaturated fatty acids in quaking animals. We suggest from these findings that myelin in the quaking mouse has certain compositional similarities with juvenile myelin, but it may be an abnormal type of myelin.     

In vitro acylation of myelin PLP and DM-20 in the quaking mouse brain

Both proteolipid proteins (PLP) and DM-20 were found to be present by the immunoblot technique in myelin isolated from quaking mouse brain; however, the relative concentration of these proteins in myelin from quaking brain was substantially reduced when compared to the control. Brain slices from littermate control and quaking mice were incubated with [³H]palmitic acid to determine the incorporation of fatty acid into myelin proteolipid proteins. Fluorography of gels containing myelin proteins from control and quaking mice brain revealed that both PLP and DM-20 were acylated. The incorporation of [³H]palmitic acid into quaking myelin PLP and DM-20 was reduced by 75% and 20% respectively of those in control brain. The significance of differential acylation of quaking myelin PLP and DM-20 is discussed with respect to availability of non-acylated pools of proteolipid proteins and the activities of acylating enzymes.     

Developmental Expression of the Myelin Proteolipid Protein and Basic Protein mRNAs in Normal and Dysmyelinating Mutant Mice

Expression of the myelin proteolipid protein (PLP) was examined in the nuclei and polysomes of 12-27-day-old quaking, jimpy, and shiverer mouse brains and in 2-27-day-old normal brains and compared with expression of the myelin basic proteins (MBPs). Northern blots showed the presence of multiple mouse PLP RNAs, the developmental expression of which coincided with myelination. Two major mouse PLP RNAs, 3.5 and 2.6 kilobases in length, were observed in both cytoplasmic polyribosomes and nuclei, and, in addition, a larger 4.6-kilobase PLP RNA was observed in nuclei. Quantitative measurements with slot blot analyses showed that the levels of PLP and MBP RNAs peaked simultaneously at 18 days in nuclei but that maximal levels of PLP RNA lagged behind MBP RNA by several days in the polysomes. The developmental expression of both major classes of myelin protein mRNAs was affected in all three mutants. In shiverer brains, the levels of PLP mRNA in polysomes and nuclei were only 30-55% of control levels after 15 days. Thus, the deletion of a portion of the MBP gene appeared to have a major effect on the expression of the PLP gene in this mutant. In jimpy mice, where the mutation has been shown to involve the PLP gene, expression of MBP mRNA was also severely reduced, to less than 25% of control values. In quaking brains, the expression of each gene followed its own developmental course, different from each other and different from the normal mouse. The extent to which the expression of PLP and MBP was affected by the quaking mutation depended on the age at which it was examined.     

Precise nucleotide location of the 5' ends of RNA-primed nascent light strands of mouse mitochondrial DNA

The mouse mitochondrial DNA origin of light-strand replication has been defined as a 32-nucleotide region located among five transfer RNA genes in the genomic sequence. A distinctive feature of this origin is its potential to form a perfectly complementary stem and 11-nucleotide loop structure. Previous studies have demonstrated that the 5′ ends of nascent light strands map within this region and a major trinucleotide ribosubstitution site in closed circular mouse mitochondrial DNA has been mapped within the stem sequence.Direct analysis and precise localization of the 5′ ends of nascent light strands indicate that essentially all 5′ ends are ribonucleotides mapping in the originspecific dyadic structure. The major 5′ end identified is the rG at position 5187 in the genomic sequence. Priming of replication most likely occurs within the loop portion of the potential dyad and continues for 2 to 16 nucleotides with a sharply defined switch to deoxyribonucleotide synthesis. This functional transition point is identical in map position to the trinucleotide ribosubstitution site in mature, closed circular mitochondrial DNA.     

Quaking is essential for blood vessel development

For nearly 40 years functional studies of the mouse quaking gene (qkI) have focused on its role in the postnatal central nervous system during myelination. However, the homozygous lethality of a number of ENU-induced alleles reveals that quaking has a critical role in embryonic development prior to the start of myelination. In this article, we show that quaking has a previously unsuspected and essential role in blood vessel development. Interestingly, we found that quaking, a nonsecreted protein, is expressed in the yolk sac endoderm, adjacent to the mesodermal site of developing blood islands, where the differentiation of blood and endothelial cells first occurs. Antibodies against PE-CAM-1, TIE-2 and SM-alpha-actin reveal that embryos homozygous for the qk(k2) allele have defective yolk sac vascular remodeling and abnormal vessels in the embryo proper at midgestation, coinciding with the timing of embryonic death. However, these mutants exhibit normal expression of Nkx2.5 and alpha-sarcomeric actin, indicating that cardiac muscle differentiation was normal. Further, they had normal embryonic heart rates in culture, suggesting that cardiac function was not compromised at this stage of embryonic development. Together, these results suggest that quaking plays an essential role in vascular development and that the blood vessel defects are the cause of embryonic death.     

Messenger RNAs from the transforming region of bovine papilloma virus type I

Messenger RNAs present in C127 mouse cells transformed by bovine papilloma virus type 1 (BPV-1) were studied by the S1 nuclease protection technique, Northern blotting, and electron microscopic heteroduplex analysis. The results revealed at least five classes of spliced mRNAs which we designate types 1 to 5. They had a common poly(A) addition site located at co-ordinate 53 and all mRNAs, except the type 3 mRNAs, contained an exon located between co-ordinates 41 and 53. In the type 1 mRNAs this exon was connected to a very short leader sequence located around co-ordinate 31. The type 2 mRNAs contained 220 to 400-nucleotide long leaders which were located approximately 1.5 X 10(3) base-pairs further upstream. Two different subclasses of type 2 molecules (2A and 2B) were identified and these had slightly different leaders. The type 4 mRNAs contained a bipartite leader, whereas the type 5 mRNAs carried an approximately 900-nucleotide long leader. The type 3 mRNAs consisted of a main exon located between co-ordinates 32 and 53, linked to the same leader as is present in the type 2A mRNAs. A cap site which presumably is utilized by the type 2A, type 3, type 4 and type 5 mRNAs was mapped at nucleotide 89 in the BPV-1 sequence. A putative cap site for the type 1 mRNAs was mapped at co-ordinate 31.     

cDNA cloning and amino acid sequence for human myelin-associated glycoprotein

cDNA clones of human myelin-associated glycoprotein were isolated and analyzed. The combination of the two overlapping cDNA clones covered the full coding region and the complete amino acid sequence was deduced. In rat and mouse, expression of the two forms of mRNA is developmentally regulated; the mRNA without exon 12 portion is expressed mainly in the actively myelinating stage of development. Although the cDNA library used here was prepared from adult human brain poly(A)+ RNA, all five clones obtained corresponded to the mRNA without exon 12 portion.     

A Short Sequence within Two Purine-Rich Enhancers Determines 5′ Splice Site Specificity

Purine-rich enhancers are exon sequences that promote inclusion of alternative exons, usually via activation of weak upstream 3′ splice sites. A recently described purine-rich enhancer from the caldesmon gene has an additional activity by which it directs selection of competing 5′ splice sites within an alternative exon. In this study, we have compared the caldesmon enhancer with another purine-rich enhancer from the chicken cardiac troponin T (cTNT) gene for the ability to regulate flanking splice sites. Although similar in sequence and length, the two enhancers demonstrated strikingly different specificities towards 5′ splice site choice when placed between competing 5′ splice sites in an internal exon. The 32-nucleotide caldesmon enhancer caused effective usage of the exon-internal 5′ splice site, whereas the 30-nucleotide cTNT enhancer caused effective usage of the exon-terminal 5′ splice site. Both enhancer-mediated splicing pathways represented modulation of the default pathway in which both 5′ splice sites were utilized. Each enhancer is multipartite, consisting of two purine-rich sequences of a simple (GAR)_n repeat interdigitated with two enhancer-specific sequences. The entire enhancer was necessary for maximal splice site selectivity; however, a 5- to 7-nucleotide region from the 3′ end of each enhancer dictated splice site selectivity. Mutations that interchanged this short region of the two enhancers switched specificity. The portion of the cTNT enhancer determinative for 5′ splice site selectivity was different than that shown to be maximally important for activation of a 3′ splice site, suggesting that enhancer environment can have a major impact on activity. These results are the first indication that individual purine-rich enhancers can differentiate between flanking splice sites. Furthermore, localization of the specificity of splice site choice to a short region within both enhancers indicates that subtle differences in enhancer sequence can have profound effects on the splicing pathway.     

Alpha hydroxylation of lignoceric acid to cerebronic acid during brain development. Diminished hydroxylase activity in myelin-deficient mouse mutants.

Alpha Hydroxylation of lignoceric acid (n-tetracosanoic acid) to cerebronic acid (2-hydroxylignoceric acid) by postnuclear preparations of brains from developing rat, mouse, and several neurological mouse mutants was studied. The preparations of brains from jimpy and myelin synthesis deficiency (msd) mice were found to synthesize cerebronic acid at less than 10 percent of their control rates, and those from quaking and dilute-lethal approximately 30 and 50 percent, respectively. The apparent low rate of in vitro hydroxylation by brains of the mutant mice appeared to be due to decreased synthesis rather than increased oxidation of cerebronic acid. Mixing experiments eliminated the possibility of an inhibitor in the mutant or an activator in normal animals. The preparations of brains from wabbler-lethal, ducky, and weaver mice showed normal activity. The developmental pattern of the hydroxylase activity was examined in quaking, jimpy, and their control mice. In normal brains the hydroxylase activity was low in the immediate postnatal period, increased sharply between 10 and 20 days after birth, and fell to a low level following maturation of the brain. The hydroxylase activity in quaking mice changed similarly during brain development but at a much reduced level. The brains of jimpy mice had barely detectable hydroxylase activity which changed little with age and reached a peak at about 15 days postpartum. The subnormal hydroxylase activity in brains of quaking mice and the near absence in brains of jimpy and msd mice correlate with the observations that myelin deficiency is more severe in jimpy and msd than in quaking. These results suggest a close association of the synthesis of cerebronic acid with the synthesis of the characteristic myelin lipid that is cerebroside (N-acyl sphingosine beta-D-galactoside).