High sensitivity quantification of RNA from gels and autoradiograms with affordable optical scanning.

To increase sensitivity and to improve normalization of RNA levels in Northern blot analysis, a comparatively inexpensive optical scanner was utilized for digitizing photonegatives of ethidium bromide stained gels and autoradiograms. The optical scanner captures the image with a maximum resolution of 300 dots per inch by assigning one of 256 gray levels (8-bit) to each dot in the image. With the use of the public domain NIH Image program (requires a Macintosh II and an 8-bit video card), gel or autoradiogram bands in the digitized image are selected and their average gray scale density measured. We found that the digitized image of a photonegative of a TAE (Tris-acetate/EDTA) agarose gel, loaded incrementally with 50-1500 ng total RNA, produced a linear response over a 4-fold range down to 100 ng (R2 greater than 0.950). In utilizing "quantification" gels like this, RNA samples that are too dilute or too small for traditional spectrophotometric techniques can be normalized and loaded uniformly onto subsequent Northern gels. Results from autoradiogram scans demonstrate highly linear gray scale responses over a 4-fold range of total RNA (R2 greater than 0.950) that are reproducible with different blots and probe types (e.g., riboprobe, cDNA and oligonucleotide). In addition, we describe a normalization technique using a 30-mer oligonucleotide probe for rat 28S ribosomal RNA as a measure of total RNA loaded per gel lane. Altogether, this scanning, ribosomal RNA normalization system allows the measurement of relative changes between 20% and 400% using standard autoradiographic methods.     

Codon usage in the vertebrate hemoglobins and its implications

A study of codon usage in vertebrate hemoglobins revealed an evolutionary trend toward elevated numbers of CpG codon boundary pairs in mammalian hemoglobin alpha genes. Selection for CpG codon boundaries countering the generally observed CpG suppression is strongly suggested by these data. These observations parallel recently published experimental results that indicate that constitutive expression of the human alpha-globin gene appears to be determined by regulatory information encoded within the structural gene. The possibility is raised that, in the absence of selection, CpG decay can be used to date the evolutionary origin of a mammalian alpha pseudogene from its active alpha gene.      

Evolution of crystallins: expression of lens-specific proteins in the blind mammals mole (Talpa europaea) and mole rat (Spalax ehrenbergi)

The mole (Talpa europaea; Insectivora) and the mole rat (Spalax ehrenbergi; Rodentia) both have degenerated eyes as a convergent adaptation to subterranean life. The rudimentary eye lenses of these blind mammals no longer function in a visual process. The crystallin genes, which display a lens-specific expression pattern, were studied in these blind mammals and in related species with normal eyes by hybridizing their genomic DNAs with probes obtained from cDNA clones for alpha A-, alpha B-, and beta Bp-crystallins from calf and gamma 3- crystallin from the rat. For all crystallin genes examined, the hybridization signals of mole and mole rat genomic DNA were comparable, respectively, with those of shrew and of rat and mouse, normal-vision representatives of the orders Insectivora and Rodentia. The expression of the crystallins at the protein level was tested by using antiserum specific for alpha-crystallin in immunofluorescence reactions on lens sections of mole and mole rat eyes and by using antisera against the beta- and gamma-crystallins on sections of the mole eye. All antisera gave positive fluorescence reactions exclusively with lens tissue of these blind mammals, indicating that the crystallins are still normally expressed despite the fact that these lenses have had no function in a visual process in these mammals for at least many million years. These findings apparently imply that some unknown selective advantage has conserved the crystallin genes and their expression after the loss of normal function of the lenses.      

The evolutionary history of the amylase multigene family in Drosophila pseudoobscura

In Drosophila pseudoobscura, the amylase (Amy) multigene family is contained within a series of inversions, or gene arrangements, on the third chromosome. The Standard (ST), Santa Cruz (SC), and Tree Line (TL) inversions are central to the phylogeny of arrangements, and have clusters of other arrangements derived from them. The gene arrangements belonging to each of these three clusters have a characteristic number of Amy genes, ranging from three in ST to two in SC to one in TL. This distribution pattern can reflect a history of either duplications or deletions, although the data available in the past did not permit a decision between these alternatives. We provide unambiguous evidence that three Amy genes were present before the divergence of the ST, SC, and TL arrangements. Thus, the current status of the Amy multigene family is the result of deletions in the TL and SC arrangements, which created three new pseudogenes: TL Amy2-psi, TL Amy3-psi, and SC Amy3- psi. Analysis of pseudogene sequences revealed that, in the SC and ST arrangements, pseudogene evolution has been retarded, most likely due to the homogenization effect of gene conversion. Finally, by determining the original copy number, we have reconstructed the evolutionary history of the Amy multigene family and linked it with the evolution of the central gene arrangements.      

Evidence for gene conversion in the amylase multigene family of Drosophila pseudoobscura

The alpha-amylase (Amy) multigene family in Drosophila pseudoobscura is located on the third chromosome, which is polymorphic for more than 40 inverted gene arrangements. The number of copies in this family ranges from one to three, depending on the arrangement in question. A previous study of the three Amy genes from the Standard (ST) arrangement suggested either that duplicated copies (Amy2 and Amy3) are functionally constrained or that they are undergoing gene conversion with Amy1. In order to elucidate further the pattern of molecular evolution in this family, we cloned and sequenced four additional Amy genes, two from the Santa Cruz (SC) and two from the Chiricahua (CH) gene arrangement. Of the two alternatives, only the hypothesis of gene conversion is supported by the sequence analysis. The homogenization effect of gene conversion has been strongest in SC, whose copies differ by only two nucleotides, less noticeable in ST, and negligible in the CH. Furthermore, the action of gene conversion is apparently localized, occurring only in the coding region. Interestingly, these results concur with the findings of other workers for the duplicated Amy genes in the Drosophila melanogaster group. Thus, the occurrence of gene conversion in the Amy multigene family seems to be a common feature in the Drosophila species studied so far.      

Mapping of multiple binding domains of the superantigen staphylococcal enterotoxin A for HLA.

Multiple binding sites on the staphylococcal enterotoxin A (SEA) molecule which interact with class II MHC Ag have been suggested by previous studies comparing SEA binding with that of another superantigen, toxic shock syndrome toxin-1. Using the synthetic peptide approach we have identified multiple regions of the SEA molecule which are responsible for binding to HLA Ag on Raji cells. Overlapping peptides were synthesized corresponding to the complete amino acid sequence of SEA: SEA(1-45), SEA(39-66), SEA(62-86), SEA(83-104), SEA(102-124), SEA(121-149), SEA(146-173), SEA(166-193), SEA(187-217), and SEA(211-233). Like the native SEA molecule, all of the peptides exhibited relatively high beta-sheet and low alpha-helical structure as determined by circular dichroism spectroscopy. A direct competition assay was employed with peptide blockage of 125I-SEA binding to MHC Ag. SEA(1-45), SEA(39-66), SEA(62-86), and SEA(121-149) but none of the other peptides blocked binding to Raji cells. The relative potency of the peptides in blocking SEA binding was determined with SEA(39-66) much greater than SEA(1-45) = SEA(62-86) = SEA(121-149). Peptide competition was seen at concentrations as low as 55 microM. Further, antibodies were produced to all of the peptides and tested for their ability to bind to SEA and inhibit SEA binding to HLA. Consistent with the direct inhibition of binding, antisera to SEA(1-45), SEA(39-66), and SEA(62-86) reduced the ability of SEA to bind Raji cells, whereas, antisera to the remaining peptides failed to block binding. The data suggest that the binding of the superantigen SEA to MHC molecules involves several N-terminal regions on SEA as well as an additional internal domain. This allows for the presence of multiple binding sites in an extended N-terminal region of the SEA molecule or a discontinuous binding epitope.     

Assessment of inter- and intra-population variation in quinoline alkaloid profile of juvenile shoot cultures of Cinchona ledgeriana: effect of method of nutrient delivery on alkaloid profile

Intra-population quinoline alkaloid profiles surveying quinine, quinidine, cinchonine and cinchonidine were determined for each of five populations of Cinchona ledgeriana grown as shoot-culture for 125 days. No significant difference in respect of mean alkaloid content between populations was detected. In contrast, there was considerable between-seedling variation in alkaloid content within each population. When nutrients were delivered to shoot-cultures in droplet form by means of an aerosol spray (as compared to the supply of nutrients direct from agar-or liquid-based reservoirs) alkaloid profile was greatly perturbed; most notable in this respect was a four-fold increase in the production of cinchonidine concomitant with a four-fold decrease in the production of cinchonine. These data are discussed with reference to the optimisation of quinoline alkaloid production by juvenile shoot-cultures of Cinchona ledgeriana.     

Use of pattern recognition to classify beef cardiovascular tissues on the basis of their trace metal compositions.

The pattern recognition procedure of discriminant analysis has been used to characterize the trace metal profiles created by the concentrations of 8 trace metals in 15 anatomic sites of beef heart tissue. Metals analyzed were copper, tin, lead, molybdenum, strontium, cesium, barium, and aluminum. Anatomic sites sampled included main pulmonary artery, aorta, mitral and tricuspid valves, left and right coronary arteries, os cordis, right atrium, left atrial appendage, crista supraventricularis, left bundle branch, free wall of the right and left ventricles, interventricular septum, and papillary muscle of the left ventricle. The striking features of the data were: (1) All specimens of the mitral valve, tricuspid valve, and os cordis were ambiguously described by their trace metal profiles; (2) the four blood vessels constituted two groups of two tissues each (aorta, main pulmonary artery; left and right coronary arteries); (3) tissues derived from ordinary and specialized myocardium were quite different from blood vessels, heart valves and os cordis. Using these profiles, 85% of the specimens analyzed were correctly classified by discriminant analysis with respect to their anatomic origin.