Expression of myelin proteins in the developing human spinal cord: Cloning and sequencing of human proteolipid protein cDNA

Abstract

A full-length clone for the human proteolipid protein (PLP) was isolated from a cDNA library constructed from poly(A)+ RNA isolated from fetal spinal cords obtained at 15-24 weeks of conceptional age. The sequence of the human PLP cDNA was determined, and the deduced amino acid sequence was found to be identical with that of rat PLP. Comparison of human and rat PLP cDNA clones indicated that the coding regions retained 97% homology and that there were also other areas of conserved sequence. The human 5'-untranslated region was 93% homologous to that of the rat. The 3'-untranslated region was, overall, 73% homologous to that of the rat with areas containing greater than 84% homology in the first 400 and last 200 nucleotides. The most variability within the 3'-untranslated region occurred between nucleotides 2,000-2,500, where homology with the rat cDNA dropped to 55%. Expression of PLP in the human spinal cord between 11 and 23 weeks after conception was examined and compared with the expression of the myelin basic protein (MBP). RNA was isolated from pooled human spinal cords obtained at three periods of development: 11-14 weeks, 17-19 weeks, and 21-23 weeks. Northern blot analysis revealed a 3.2-kilobase (kb) PLP mRNA that was present at higher abundance in the 21-23-week spinal cord RNA than in the 17-19-week or the 11-14-week samples. The 17-19-week RNA sample also contained a PLP-hybridizing band at 2.2 kb which may possibly have arisen by utilization of alternative polyadenylation signals. Messenger RNA for MBP was detectable at 11-14 weeks but was readily evident in both the 17-19- and 21-23-week age groups. Immunoblot analysis of whole spinal cord homogenates indicated that polypeptides for MBP preceded the appearance polypeptides for PLP by 3-4 weeks.

Origin of Oligodendrocytes within the Human Spinal Cord

“…DM20 transcripts and protein were already detected in human spinal cord at 18 WG by Northern and Western analysis (Kronquist et al, 1987). Here we detected by in situ hybridization PLP/DM20 transcripts in the VZ region of the neural tube at 30 dpc and in the ventral VZ of the spinal cord at 45 dpc.…”

The human myelin basic protein gene is included within a 179-kilobase transcription unit: expression in the immune and central nervous systems.

“…Two bands at 5.1 kb and 2.6 kb were detected in each sample. Expression of the two mRNAs in the spinal cord was expected because at this age of fetal development, myelina- tion is commencing (11,12). Further, this developmental tected in the human fetal thymus sample.…”

“…Exons 2 and 5 of the MBP gene are alternatively spliced to produce four MBP mRNAs encoding polypeptides that range in size from 17 to 21.5 kDa (10,11). These MBP mRNAs and proteins begin to be expressed in the developing human fetal spinal cord between 10 and 20 weeks after conception (12).…”

Expression of Myelin Protein Genes and Other Myelin Components in an Oligodendrocytic Cell Line Conditionally Immortalized with a Temperature‐Sensitive Retrovirus

“…10, N20.1 cells grown at 34°C alternatively spliced the PLP primary transcript to produce both PLP and DM20 mRNAs. However, the predominant alternatively spliced transcript was that of the DM20 mRNA, which is the splicing pattern exhibited by oligodendrocytes in vivo during the early stages of the expression of the PLP gene (Kronquist et al, 1987;Gardinier and Macklin, 1988;Schindler et al, 1990;Timsit et al, 1992). No difference in the relative ratios of PLP to DM20 were observed when the cells were grown at 39°C for up to 37 days.…”