Levels of nerve growth factor and neurotrophin-3 are affected differentially by the presence of p75 in sympathetic neurons in vivo.

The development and survival of sympathetic neurons is critically dependent on the related neurotrophic factors nerve growth factor (NGF) and neurotrophin-3 (NT3), the actions of which must be executed appropriately despite spatial and temporal overlaps in their activities. The tyrosine receptor kinases, trkA and trkC, are the cognate receptors for NGF and NT3, respectively. The p75 neurotrophin receptor has been implicated in neurotrophin binding and signaling for both NGF and NT3. In this study, the authors used mice that overexpressed NGF (NGF-OE) or NT3 (NT3-OE) in skin and mice that lacked p75 (p75(-/-)) to understand the dynamics of sympathetic neuron response to each neurotrophin and to address the role of p75. NGF and NT3 were measured in sympathetic ganglia and skin (a major target of sympathetic neurons) by using the enzyme-linked immunosorbent assay (ELISA) technique. A three- to four-fold increase in skin NT3 was seen in both NT3-OE and p75(-/-) mice. Moreover, both mouse lines exhibited a three-fold increase in ganglionic NT3. However, the increase in ganglionic NT3 was accompanied by a decrease in ganglionic NGF in p75(-/-) mice but not in NT3-OE mice. This indicated that p75 plays an important role in determining the level of NGF within sympathetic neurons. In NGF-OE mice, the overexpression of NGF was correlated with increased ganglionic NGF and increased ganglionic expression of p75 mRNA. In addition, in NGF-OE mice, ganglionic trkC expression was decreased, as was the amount of NT3 present within sympathetic ganglia. These results indicate that the level of p75 is integral in determining the level of sympathetic NGF and that NGF competes with NT3 by increasing the expression of p75 and decreasing the expression of trkC.

Effects of mutation of the Olf-1 motif on transgene expression in olfactory receptor neurons.

We have examined the effect of mutating the Olf-1 binding motif of the olfactory marker protein (OMP) promoter in determining olfactory neuron-specific gene expression in adult tissues and during embryonic development. The proximal Olf-1 motif located 170 nucleotides upstream of the transcription start site of the OMP gene was mutated to prevent its interaction with the Olf-1 factor in vitro. The wild-type and mutated fragments of the OMP gene extending from -239 to +55 nucleotides relative to the transcription start site were used to direct expression of a lacZ reporter gene in transgenic mice. The transgenic animals were analyzed for cell-specific and developmental expression of the reporter gene. We demonstrate that the mutation that prevents interaction of Olf-1 with its binding site does not alter the temporal and spatial patterns of gene expression in olfactory sensory neurons but does alter the specificity and level of expression in other neuronal populations. These observations are consistent with our demonstration that the mutated Olf-1 site interacts with nuclear proteins present in the central nervous system (CNS).

Rat kidney band 3 Cl-/HCO3- exchanger mRNA is transcribed from an alternative promoter.

We have previously shown that the rat kidney band 3 Cl-/HCO3- exchanger mRNA encodes an NH2-terminal truncated form of band 3 and that its 5' end differs from that of the erythrocyte band 3 mRNA (K. E. Kudrycki and G. E. Shull. J. Biol. Chem. 264: 8185-8192, 1989). To determine the genetic basis for the alternative kidney and erythroid mRNAs, we 1) isolated and characterized a rat erythroid band 3 cDNA, 2) isolated the rat band 3 gene and determined the exon/intron organization of sequences corresponding to the alternative 5' ends of the rat kidney and erythroid mRNAs, and 3) identified the transcription initiation sites of the two transcripts. The unique sequences at the 5' end of the rat erythroid mRNA are derived from exons 1-3 and are followed directly by sequences from exon 4 that are common to both mRNAs. In the kidney mRNA, sequences upstream of exon 4 are derived entirely from intron 3. Primer extension and S1 nuclease protection analyses demonstrate the presence of multiple transcription initiation sites for the rat erythroid band 3 mRNA at the beginning of exon 1, whereas the transcription initiation site for the kidney mRNA is located within intron 3. Thus two distinct promoters, separated by almost 5 kb of genomic sequence, are responsible for the highly tissue-specific transcription of the alternative rat erythroid and kidney band 3 mRNAs.

The predicted translation product of a cardiac AE3 mRNA contains an N terminus distinct from that of the brain AE3 Cl-/HCO3- exchanger. Cloning of a cardiac AE3 cDNA, organization of the AE3 gene, and identification of an alternative transcription initiation site.

We have isolated cDNAs encoding a cardiac variant of the AE3 Cl-/HCO3- exchanger from a rat heart library. The predicted cardiac AE3 polypeptide is 1030 amino acids in length, while the AE3 variant expressed in brain consists of 1227 amino acids. The C-terminal 957 amino acids of both polypeptides are identical, but the cardiac protein contains a unique N-terminal sequence of 73 amino acids which replaces the first 270 amino acids of the brain form. To determine the genetic basis for the differences between the cardiac and brain AE3 variants, we isolated and characterized the rat gene. Exons 1-6 contain the 5'-untranslated sequence and the first 270 codons of the brain mRNA, while exons 7-23 contain the coding and 3'-untranslated sequences which are common to the brain and cardiac mRNAs. The 5'-untranslated and unique N-terminal coding sequences of the cardiac mRNA are contained within a single exon, termed C1, which forms part of the intron between exons 6 and 7 of the brain transcription unit. Primer extension and S1 nuclease protection assays demonstrate that transcription of the cardiac AE3 mRNA precursor is intiaited within this intron. Therefore, alternative promoter and exon usage are involved in generation of the two AE3 mRNAs.

cDNA cloning and tissue distribution of mRNAs for two proteins that are related to the band 3 Cl-/HCO3- exchanger.

Complementary DNAs encoding two proteins that are related to the Band 3 Cl-/HCO3- exchanger, designated B3RP2 and B3RP3, have been isolated from rat stomach, brain, and kidney libraries. B3RP2 is 1234 amino acids in length and has an Mr of 136,644. B3RP3 is 1227 amino acids in length and has an Mr of 135,405. B3RP2 and B3RP3 exhibit 52 and 50% amino acid identity to Band 3, respectively, and 56% identity to each other. The N-terminal cytoplasmic regions of B3RP2 and B3RP3, which span about 700 amino acids, are more extensive than that of Band 3. The C-terminal hydrophobic regions of the three proteins exhibit a high degree of amino acid identity (64-69%) and have very similar hydropathy profiles, suggesting that they have the same transmembrane organization. The tissue distribution of mRNAs encoding B3RP2, B3RP3, and the Band 3 Cl-/HCO3- exchanger were examined by Northern blot hybridization using poly(A)+ RNAs from a broad range of muscle and non-muscle tissues and from sections of the gastrointestinal tract. B3RP2 mRNAs are expressed in all tissues examined. The highest levels, which include at least three different transcripts, occur in stomach. B3RP3 mRNAs, which also consist of several different transcripts, have a more limited tissue distribution. The highest levels occur in heart, and in gastrointestinal sections the highest levels are in the forestomach. Band 3 mRNAs were observed in many tissues but high levels of expression occurred only in spleen and kidney. Five Band 3 transcripts, ranging in size from 3.6 to 4.9 kilobases, were detected, including three that are expressed in heart.

Primary structure of the rat kidney band 3 anion exchange protein deduced from a cDNA.

A rat kidney cDNA library was screened using an oligonucleotide hybridization probe derived from an amino acid sequence of the mouse erythrocyte Band 3 Cl-/HCO3- exchanger. Ten cDNAs representing a single class of mRNA and encoding the kidney Band 3 anion exchanger were isolated. Analysis of the deduced amino acid sequence revealed that kidney Band 3 is a truncated version of erythrocyte Band 3. The 848-amino acid rat kidney protein is virtually identical to mouse erythrocyte Band 3 except that it lacks the first 79 amino acids of the N-terminal cytoplasmic domain. Exons 1, 2, and 3 of the Band 3 gene, which are included in erythrocyte Band 3 mRNA, are not present in the kidney cDNA. The 5'-untranslated regions of kidney Band 3 transcripts contain sequences from the third intron and exons 4 and 5 of the Band 3 gene. The sequences from exons 4 and 5 that serve as untranslated sequences in the rat kidney mRNA have the potential to encode amino acids 46-79 of erythrocyte Band 3 but are not part of an open reading frame; the apparent translation initiation site corresponds to codon 80 of mouse erythrocyte Band 3 mRNA. Northern blot hybridization studies indicate that two additional Band 3 mRNAs, which differ in size from the major 4.5-kilobase pair transcript in kidney, are expressed at low levels in kidney and brain.