Lori L. Hampton

Identification of a signaling network in lateral nucleus of amygdala important for inhibiting memory specifically related to learned fear.

We identified the Grp gene, encoding gastrin-releasing peptide, as being highly expressed both in the lateral nucleus of the amygdala, the nucleus where associations for Pavlovian learned fear are formed, and in the regions that convey fearful auditory information to the lateral nucleus. Moreover, we found that GRP receptor (GRPR) is expressed in GABAergic interneurons of the lateral nucleus. GRP excites these interneurons and increases their inhibition of principal neurons. GRPR-deficient mice showed decreased inhibition of principal neurons by the interneurons, enhanced long-term potentiation (LTP), and greater and more persistent long-term fear memory. By contrast, these mice performed normally in hippocampus-dependent Morris maze. These experiments provide genetic evidence that GRP and its neural circuitry operate as a negative feedback regulating fear and establish a causal relationship between Grpr gene expression, LTP, and amygdala-dependent memory for fear.

Dominant and recessive deafness caused by mutations of a novel gene, TMC1, required for cochlear hair-cell function.

Positional cloning of hereditary deafness genes is a direct approach to identify molecules and mechanisms underlying auditory function. Here we report a locus for dominant deafness, DFNA36, which maps to human chromosome 9q13–21 in a region overlapping the DFNB7/B11 locus for recessive deafness. We identified eight mutations in a new gene, transmembrane cochlear-expressed gene 1 (TMC1), in a DFNA36 family and eleven DFNB7/B11 families. We detected a 1.6-kb genomic deletion encompassing exon 14 of Tmc1 in the recessive deafness (dn) mouse mutant, which lacks auditory responses and has hair-cell degeneration. TMC1 and TMC2 on chromosome 20p13 are members of a gene family predicted to encode transmembrane proteins. Tmc1 mRNA is expressed in hair cells of the postnatal mouse cochlea and vestibular end organs and is required for normal function of cochlear hair cells.

Mutations of ESPN cause autosomal recessive deafness and vestibular dysfunction

We mapped a human deafness locus DFNB36 to chromosome 1p36.3 in two consanguineous families segregating recessively inherited deafness and vestibular areflexia. This phenotype co-segregates with either of two frameshift mutations, 1988delAGAG and 2469delGTCA, in ESPN, which encodes a calcium-insensitive actin-bundling protein called espin. A recessive mutation of ESPN is known to cause hearing loss and vestibular dysfunction in the jerker mouse. Our results establish espin as an essential protein for hearing and vestibular function in humans. The abnormal vestibular phenotype associated with ESPN mutations will be a useful clinical marker for refining the differential diagnosis of non-syndromic deafness.

The human gastrin-releasing peptide receptor gene structure, its tissue expression and promoter☆

The human gastrin-releasing peptide receptor (hGRP-R) is aberrantly expressed in cancers of the colon, lung and prostate and mediates signals of cellular proliferation. However, the underlying mechanisms of aberrant and/or activation of hGRP-R expression are unknown. Therefore, a genomic clone is identified, the hGRP-R gene is characterized, and the hGRP-R promoter is defined. The protein coding region is divided into three exons and exon/intron splice sites occur in the proximal 2nd and distal 3rd intracellular loops of the receptor molecule. The hGRP-R locus extends over more than 27 kb and is assigned to the chromosomal band Xp22 by fluorescence in situ hybridization. With primer extension experiments, we demonstrate two major transcription start sites in gastrointestinal and breast cancer cells, located 43 and 36 bp downstream of a TTTAAA motif which is identified 407 to 402 bp upstream of the ATG start codon. The hGRP-R is found most abundantly expressed in the normal human pancreas, where four gene-specific transcripts can be detected by Northern blot analysis, whereas only two transcripts are detected in the human stomach and, very weakly, in the adrenal cortex and the brain. In contrast, the human GRP-R is not expressed in the normal human colon, lung, and prostate. Steady state hGRP-R mRNA can also be detected in some cultured cells from breast, lung, and duodenal cancer. Robust hGRP-R promoter activity is demonstrated in a duodenal carcinoma cell line that natively expresses the functional hGRP-R. Truncation studies suggest a CRE motif, located 112 bp upstream of the major transcription start site, is required to confer basal hGRP-R promoter activity in duodenal cancer cells. These studies provide the necessary data to further elucidate molecular mechanisms of aberrant hGRP-R expression in human cancers.

A human gene encodes a putative G protein-coupled receptor highly expressed in the central nervous system

The mammalian bombesin (Bn)-like neuropeptide receptors gastrin-releasing peptide receptor (GRP-R) and neuromedin B receptor (NMB-R) transduce a variety of physiological signals that regulate secretion, growth, muscle contraction, chemotaxis and neuromodulation. We have used reverse transcription-polymerase chain reaction (PCR) to isolate a cDNA from human brain mRNA, GPCR/CNS, that encodes a putative G protein-coupled receptor (GPCR) based upon the presence of the paradigmatic seven heptahelical transmembrane domains in its predicted amino acid sequence. Analysis of the deduced protein sequence of GPCR/CNS reveals this putative receptor to be 98% identical to the deduced amino acid sequence of a recently reported gene product and minimally identical (approximately 23%) to both murine GRP-R and human endothelin-B (ET-B) receptor. Our deduced protein sequence differs at 12 positions, scattered throughout the open reading frame, relative to the original sequence. A 3.7 kb GPCR/CNS mRNA species is expressed in vivo in a tissue-specific manner, with highest levels detected in brain and spinal cord, lower levels found in testis, placenta and liver, but no detectable expression observed in any other tissue. Analysis of GPCR/CNS genomic clones reveals that the human gene contains one intron that is about 21 kb in length that divides the coding region into two exons and maps to human chromosome 7q31. No specific binding is observed with either a newly identified ligand (DTyr6, beta Ala11, Phe13, Nle14]Bn-(6-14)) having high affinity for all Bn receptor subtypes or Bn after GPCR/CNS is stably expressed in fibroblasts. No elevation in inositol trisphosphate is observed after the application of micromolar levels of either DPhe6, beta Ala11, Phe13, Nle14]Bn-(6-14) or Bn, a concentration of agonist known to activate all four known Bn receptor subtypes. When GPCR/CNS is expressed in Xenopus oocytes, no activation of the calcium-dependent chloride channel is detected despite the addition of micromolar levels of Bn peptide agonists. We conclude that the natural ligand for this receptor is none of the known naturally occurring Bn-like peptides and the true agonist for GPCR/CNS remains to be elucidated.

A new spontaneous mutation in the mouse Ames waltzer gene, Pcdh15

A recessive deafness mutation in the mouse arose spontaneously and was identified in a colony segregating a null allele of the gastrin-releasing peptide receptor (Grpr) locus. Auditory-evoked brain stem response measurements revealed deafness in 7-week-old affected mice. By linkage analyses, the mutant phenotype was mapped near marker D10Mit186 and the protocadherin gene Pcdh15. As shown by complementation testing, the new mutation is allelic with Ames waltzer (Pcdh15(av)). Sequencing mutant-derived brain Pcdh15 cDNAs identified the insertion of a cytosine residue at nucleotide position c2099 (2099insC), which results in a frame-shift and premature stop codon. Abnormal stereocilia on inner and outer hair cells of the organ of Corti were identified by scanning electron microscopy as early as postnatal day 0 and cross-sectional histology revealed severe neuroepithelial degeneration in cochleas of 30-50-day-old mutants. The new allele of Ames waltzer, designated Pcdh15(av-Jfb), may aid in studying the histopathology associated with Usher syndrome type 1F, which is caused by a functional null allele of PCDH15.

Structure and chromosomal localization of the mouse bombesin receptor subtype 3 gene

Bombesin (BN)-like peptides/neurotransmitters mediate a broad range of physiological funtions in the gastrointestinal tract and the central nervous system through binding to their specific, high-affinity mammalian bombesin receptors. This family of heptahelical, G-protein coupled receptors includes the gastrin-releasing peptide receptor (GRP-R, or bb2), neuromedin B receptor (NMB-R, or bb1), and the bombesin receptor subtype 3 (BRS-3, or bb3). The tissue distribution of BRS-3 is quite dissimilar compared to the other two BN receptors, GRP-R and NMB-R, and a natural ligand for BRS-3 is currently unknown. Nothing is known about mechanisms regulating BRS-3 gene expression and possible association with disease. To gain insight into the underlying structure and chromosomal localization of the BRS-3 genes, bacteriophage P1 genomic clones, harboring the genes for the human and mouse BRS-3, respectively, were isolated and their structure and chromosomal localizations determined. The protein-coding region of both genes is divided into three exons and spans approximately 5kb. The loci of the BRS-3 genes were mapped to a syntenic region of the human (Xq25) and mouse (XA7.1-7.2) X-chromosome, respectively. The structural data of the BRS-3 genes derived from this study will permit future investigations of the mechanisms regulating their expression.

Exclusion of the gastrin-releasing peptide receptor (GRPR) locus as a candidate gene for Rett syndrome.

The gene for the gastrin-releasing peptide receptor (GRPR) has been mapped to a candidate region for Rett syndrome (RTT) on the short arm of the X chromosome. The recent report of a translocation that disrupted the gene in an individual with mental retardation and autistic behavior prompted us to examine GRPR as a possible locus for RTT. Genomic polymerase chain reaction amplification of exons followed by single-strand conformation analysis screening in 25 unrelated RTT-affected individuals and by direct sequencing in 12 others has failed to detect any mutation. No gross structural rearrangements were found by Southern analysis of DNA from six unrelated RTT-affected individuals. A high-frequency biallelic polymorphism caused by two single nucleotide substitutions in exon 2 was discovered. The allele frequencies were identical in the RTT population as compared to 100 normal control X chromosomes. This polymorphism will enable future evaluation of the GRPR locus as a candidate for other X-linked mental retardation or neurobehavioral syndromes.

Cloning and characterization of the Drosophila melanogaster CDK5 homolog

The D. melanogaster homolog of mammalian CDK5 has been cloned and its chromosomal location determined. The gene for Cdk5 consists of 4 exons separated by 3 short introns ranging in size from 61–160 bp. Northern blot analysis revealed a single mRNA of approximately 1.6 kb that is expressed at highest levels in the adult fly. The putative amino acid sequence for Drosophila Cdk5 predicts a protein with a mass of approximately 32 kDa that is 77% identical to its mammalian counter‐parts. Drosophila Cdk5 gene is located in polytene chromosomal region 52BC of the right arm of chromosome 2. This study provides the framework for a molecular genetic analysis of CDK5 function.

Constitutive over-expression of transforming growth factor-alpha in rat liver epithelial cells leads to increased cell cycling without transformation

SummaryOver-expression of transforming growth factor-alpha (TGF-α) is consistently seen in spontaneous transformants of rat liver derived epithelial cells (RLE Φ13) and has been implicated in the transformation of other cultured cells. We have constitutively over-expressed TGF-α in RLE Φ13 cells, which are known to express epidermal growth factor receptors, to determine if TGF-α over-expression plays a role in transformation or differentiation, or both, of these cells. Early passage RLE Φ13 cells were infected with a replication-defective murine retrovirus that expresses both the full length coding sequence for human TGF-α and the neomycin-resistance gene. Integration of the transcriptionally active provirus and expression of TGF-α mRNA were confirmed. Neither morphologic transformation nor molecular evidence for differentiation was noted in TGF-α-producing clones. However, these clones did exhibit an accelerated growth rate, increased expression of several cell cycle related genes including mitotic cyclie B1, proliferating cell nuclear antigen, c-myc, and p53 as well as increased expression of the preneoplastic marker enzyme, glutathione-S-transferase. This suggests that over-expression of TGF-α results in increased cell cycling, and that subsequent events must be necessary for cellular transformation or differentiation or both.