In many vertebrates, UV-sensitive photoreceptors have been identified by microspectrophotometry and UV-visual sensitivity has been identified by behavioral studies, but as yet no vertebrate UV-sensitive pigment gene has been isolated. We have sequenced a cDNA clone that hybridizes to short single cone cells in the zebrafish (Brachydanio rerio). These cells, which make up 25% of the cone population in zebrafish retinae, are UV-sensitive (lambda max approximately 360 nm). The visual pigment encoded by this gene is unusual in that its amino acid sequence is more homologous to the rod pigment rhodopsin (up to 89%) than to other cone pigments (35-83%). Like all other vertebrate visual pigments, it contains a lysine residue at position 296, the presumptive retinal binding site, and a glutamate residue at position 113. However, it is unique in possessing a lysine residue at position 126, which may account for the UV-sensitivity of the pigment.

An autosomal recessive deficiency of blood coagulation factor XI (FXI) has been described in Holstein cattle. Current testing methods are unsuitable for accurately identifying carriers (heterozygotes) of the disease. To identify the molecular basis of this deficiency, a polymerase chain reaction (PCR)-based strategy was implemented to clone and sequence the bovine FXI gene (F11) from animals of different genotypes. Approximately 14 kb of genomic DNA sequence and 1.8 kb of cDNA sequence, corresponding to exon 3 through the 3'-UTR, of the bovine gene were obtained. Comparison of sequences derived from homozygous normal and deficient individuals revealed that FXI deficiency in Holsteins is associated with the insertion of a 76 bp segment [AT(A)(28)TAAAG(A)(26)GGAAATAATAATTCA] within exon 12. This insertion introduces a stop codon that results in a mature FXI protein lacking the functional protease domain encoded by exons 13, 14 and 15. Based on these data, a DNA-based diagnostic test has been developed for accurate genotyping. Using this method, the frequency of the mutated allele has been determined to be 1.2% in a contemporary population of the USA Holstein sires.

In zebrafish, the first class of cone photoreceptor to become morphologically distinct is the ultravioletsensitive short single cone, at 4 days postfertilization, whereas the last class, the red-and green-sensitive double cone, becomes distinct at 10 days postfertilization. We have examined the time course of visual pigment gene expression in zebrafish using whole-mount in situ hybridization. Within the retina, opsins may be detected as early as 40 h postfertilization with the ultraviolet and rod visual pigments being expressed before the blue-(48 h) and red-(60 h) sensitive pigments. In the pineal, red-sensitive opsin is expressed at 48 h postfertilization. Visual pigment expression provides a useful tool for investigations of early cell fate in zebrafish.

Developmental switching of hemoglobins (Hbs) occurs in most vertebrates, yet the cellular and molecular basis for this process remains elusive. The zebrafish is a new genetic and developmental system that can be used to study embryogenesis, and mutants with a variety of defects in hematopoiesis have recently been derived. To initiate our studies on Hb switching in this organism, we have characterized the globins expressed in the adult. Reversed-phase high performance liquid chromatography and mass spectrometric analyses of adult peripheral blood hemolysates showed that there are three major α globins and two β globins in circulating erythroid cells. In addition, we have isolated and characterized zebrafish adult α- and β-globin cDNA clones that encode some of these globins. High levels of α- and β-globin gene expression were detected in adult erythroid cells, whereas embryonic erythroid cells expressed little, if any, of these RNAs. We have also shown that the α- and β-globin genes are tightly linked on the same chromosome and are arrayed in a 3′-5′ to 5′-3′ configuration, respectively. The characterization of these genes and regulatory elements in this globin locus will provide insight into the process of globin gene transcription. With these reagents, future studies of Hb switching in zebrafish mutants with defective hematopoiesis will be possible.