Chromosomal organization and evolutionary conservation of the human chromosome 19q linkage group containing three DNA repair genes

A series of human-rodent somatic cell hybrids were investigated by Southern blot analysis for the presence or absence of twenty-six molecular markers and three isozyme loci from human chromosome 19. Based on the co-retention of these markers in the various independent hybrid clones containing portions of human chromosome 19 and on pulsed field mapping, chromosome 19 is divided into twenty ordered regions. The most likely marker order for the chromosome is: (LDLR, C3)-(cen-MANNB)-D19S7-PEPD-D19S9-GPI-TGF$ \beta$-(CYP2A, NCA, CGM2, BCKAD)-PSG1a-(D19S8, XRCC1)-(D19S19, ATP1A3)-(D19S37, APOC2)-CKMM-ERCC2-ERCC1-(D19S62, D19S51)-D19S6-D19S50-D19S22-(CGB, FTL)-qter.^ The region of 19q between the proximal marker D19S7 and the distal gene coding for the beta subunit of chorionic gonadotropin (CGB) is about 37 Mb in size and covers about 37 cM genetic distance. The ration of genetic to physical distance on 19q is therefore very close to the genomic average OF 1 cM/Mb. Estimates of physical distances for intervals between chromosome 19 markers were calculated using a mapping function which estimates distances based on the number of breaks in hybrid clone panels. The consensus genetic distances between individual markers (established at HBM10) were compared to these estimates of physical distances. The close agreement between the two estimates suggested that spontaneously broken hybrids are as appropriate for this type of study as radiation hybrids.^ All three DNA repair genes located on chromosome 19 were found to have homologues on Chinese hamster chromosome 9, which is hemizygous in CHO cells, providing an explanation for the apparent ease with which mutations at these loci were identified in CHO cells. Homologues of CKMM and TGF$\beta$ (from human chromosome 19q) and a mini-satellite DNA specific to the distal region of human chromosome 19q were also mapped to Chinese hamster 9. Markers from 19p did not map to this hamster chromosome. Thus the q-arm of chromosome 19, at least between the genes PEPD and ERCC1, appears to be a linkage group which is conserved intact between humans and Chinese hamsters. ^

Molecular interactions and signal transfer between sensory rhodopsin-I and its cognate transducer

SRI is unique among known photoreceptors in that it produces opposite signals depending on the color of light stimuli. Absorption of orange light (587 nm) triggers an attractant response by the cell, whereas absorption of orange light followed by near-UV light (373 run) triggers a repellent response. Using behavioral mutants that exhibit aberrant color-sensing ability, we tested a two-conformation equilibrium model, using FRET and EPR spectroscopy. The essence of the model applied to SRI-HtrI is that the complex exists in a metastable two-conformer equilibrium which is shifted in one direction by orange light absorption (producing an attractant signal) and in the opposite direction by a second UV-violet photon (producing a repellent signal). First, by FRET we found that the E-F cytoplasmic loop of SRI moves toward the RAMP domain of the HtrI transducer during the formation of the orange-light activated signaling state of the complex. This is the first localization of a change in the physical relationship between the receptor and transducer subunits of the complex and provides a structural property of the two proposed conformers that we can monitor. Second, EPR spectra of a spin label probe at this cytoplasmic position showed shifts in the dark in the mutants toward shorter or longer EF loop-RAMP distances, explaining their behavior in terms of their mutations causing pre-stimulus shifts into one or the other conformer. ^ Next, we applied a novel electrophysiological method for monitoring the directionality of proton movement during photoactivation of SRI, to investigate the process of proton transfer in the photoactive site from the chromophore to proton acceptors on both the wildtype and aberrant color-response mutants. We observed an unexpected and critical difference in the two signaling conformations of the SRI-HtrI complex. The finding is that the vectoriality (i.e. movement away or toward the cytoplasm) of the light-induced proton transfer from the chromophore to the protein is opposite in formation of the two conformations. Retinylidene proton transfer is a common critical process in rhodopsins and these results are the first to show differences in vectoriality in a rhodopsin receptor, and to demonstrate functional importance of the direction of proton transfer. ^