DNAase I sensitivities in chromatin of the Xenopus laevis somatic and oocyte 5 S DNAs.

The DNAase I sensitivities of the somatic-type 5 S DNA and oocyte-type 5 S DNA have been compared in nuclei from adult somatic tissues of Xenopus laevis. Neither of these Type III genes is expressed in mature erythrocytes and only somatic-type 5 S DNA is expressed in liver. We find that somatic-type 5 S DNA is DNAase-I-sensitive in liver nuclei and less sensitive in erythrocyte nuclei, while oocyte-type 5 S DNA is insensitive in both tissues. The DNAase I sensitivity appears to be uniform across each active somatic-type 5 S DNA repeat. Two regions slightly hypersensitive to DNAase I are found only in liver somatic-type 5 S DNA. One of these regions is within the gene, overlapping with the binding site of the transcription factor (TF III A) required for 5 S RNA synthesis. Thus, the correlation between DNAase I sensitivity and gene activity previously seen for protein-coding genes also holds for these Type III genes. Our data lead us to suggest that the fully DNAase-I-sensitive chromatin conformation on 5 S DNA requires the presence both of transcription factors and RNA polymerase.

Characterization of an evolutionarily old human alphoid DNA.

A recently isolated human alphoid DNA (in plasmid pHH550) has been sequenced and found to have an exceptionally high degree of similarity to the human alphoid consensus sequence, while its component monomers are unusually heterogeneous in sequence. In contrast to other alphoid DNAs, this DNA is found in all primates tested. Thus this may be an evolutionarily old sequence similar to the one from which other human alphoid DNAs diverged. The pHH550 sequences are found on a number of human chromosomes, including 21 and 22. On chromosome 21 most members of this new sequence group are located distal to other alphoid DNAs.

A new human alphoid-like repetitive DNA sequence.

A new tandemly repetitive sequence family, having the 170 bp basic repeat characteristic of alphoid sequences, has been identified in the human genome. Its organization in the whole genome and on chromosome 21 is different from that of any of the previously described alphoid families. Members of this new family are unusually heterogeneous in sequence, and there are a number of variant sequence classes. Some of the variant classes exist in separate genomic domains, and even on a single chromosome the members of such a class are not significantly intermixed with members of another class.

Length polymorphisms in new human collagen-like loci.

Two new collagen-like loci have been identified in the human genome which have sequence similarity to the triple-helical coding region of the pro-alpha 2(I) gene. Both loci exhibit length polymorphism due to alleles that contain deletions. The deletion at one locus is 400 bp while the deletion at the second locus is 200 bp. The second locus is on chromosome 17 and its two alleles are not in Hardy-Weinberg equilibrium. These loci are candidates for involvement in connective tissue disorders.

Improved pyeloureteral visualization using glucagon during experimental intravenous urography.

Intravenous urography is optimally performed using abdominal compression for distention of the pyelocalyceal systems. Because of real and potential difficulties with the application of compression, the authors have tested in dogs the alternative of administering glucagon as a means of improving pyelocalyceal distention and ureteral visualization. In six greyhound dogs, ureteral peristalsis transiently ceased following the intravenous injection of glucagon. Objective and coded subjective analyses showed statistically significant improvement in demonstration of collecting systems and ureters when urograms were performed with glucagon, as compared with those in which the drug was not administered. The success of glucagon in improving pyeloureteral visualization in dogs encourages its experimental use in human subjects as an alternative to abdominal compression.

Families' experiences in consenting to eye donation of a recently deceased relative.

OBJECTIVE: To explore the unique experience of families who have consented to eye donation of a recently deceased relative. DESIGN: Exploratory-descriptive. SETTING: A city in western Canada. PARTICIPANTS: Seventeen relatives of 16 eye donors who had died within the previous 3 to 12 months. RESULTS: Families were in psychologic shock after the donor's death, which inhibited their recollection of events. Nurses were found to initiate the possibility of eye donation most often, and the decision to donate was made precipitously. In several cases, it was not stated clearly on the consent form what tissue was to be removed, and whether the family had agreed only to transplantation or transplantation and research. When the tissue was used for research, families had a need to believe this would eventually benefit someone. CONCLUSION: Particular care needs to be taken when reviewing the consent form with families to ensure that it reflects how they or the donor want the tissue to be used. Families can be prepared for the eventuality that the tissue will be used for research rather than transplantation by the reinforcement that the knowledge gained will help many individuals.

Differential amino acid requirements for sporulation in Bacillus subtilis.

The amino acid requirements for sporulation were studied by use of auxotrophic mutants of Bacillus subtilis 168. Cells were grown to T(0) in medium containing the test amino acid and were then transferred to a minimal medium lacking that amino acid. Omission of leucine caused no reduction in sporulation. Omission of methionine, lysine, and phenylalanine appeared to cause reduced levels of sporulation, and sporulation was completely inhibited when isoleucine, tryptophan, and threonine were omitted. The amino acids in this third class showed a sequence of requirements, with tryptophan required earlier than isoleucine, which in turn was required earlier in the sporulation process than threonine. Isoleucine omission did not affect the early sporulation functions of extracellular protease formation or septum formation, but prevented the increased levels of protein synthesis and oxygen consumption that normally accompany early sporulation stages. Isoleucine did not appear to be metabolized to other compounds in significant amounts during sporulation. The role of isoleucine in the sporulation process remains unclear.

Quick-blot: selective mRNA or DNA immobilization from whole cells.

Quick-blot, a method for selectively immobilizing either mRNA or DNA on nitrocellulose, is described in detail. Essential elements of the procedure for immobilizing DNA include tissue lysis, proteinase K treatment, solubilization of nucleic acids in hot 12.2 molal NaI, passage through a nitrocellulose filter, and acetylation of residual protein with acetic anhydride. Advantages include speed, quantitative recovery, low background, and elimination of the usual baking step. Essential elements of the procedure for selectively immobilizing mRNA include dissolving cells in Brij-35 and desoxycholate, proteinase K treatment, solubilizing nucleic acids in room temperature 12.2 molal NaI, filtration through nitrocellulose, and acetylation of residual protein. Advantages include selective immobilization of mRNA but not tRNA, rRNA, or DNA, and the maintenance of biological activity of the immobilized mRNA. Control experiments to optimize the procedures and examples of their application are shown.

The genomic organization of dispersed tRNA and 5 S RNA genes in Xenopus laevis.

The 5 S DNAs and several tDNAs of Xenopus laevis reside primarily in large clusters of tandem repeating units. We have discovered that a substantial number of these genes, along with portions of their adjacent spacer sequences, are also located in dispersed genomic locations apart from the major clusters. This was accomplished by "null-digesting" total genomic DNA with restriction enzymes that do not cut within the X. laevis tDNA or 5 S DNA major repeats. The tDNA and 5 S DNA main clusters therefore remain intact and can be easily separated on gels from the dispersed tDNAs and 5 S DNAs present as low molecular weight restriction fragments. Probing these smaller fragments with different portions of the major repeats has revealed that many of the dispersed genes are organized differently from the corresponding tDNAs and 5 S DNAs of the primary clusters. Some of the fragments containing dispersed genes are actually present in multiple copies. In addition, many tDNA null-digestion fragments contain more than one type of tRNA coding region. One set of "dispersed" tDNAs actually comprises a tandemly arranged minor tDNA family which has retained the same repeat length (3.18 kb) as the major tDNA family, but has a substantially different organization. There is significant population polymorphism in the organization of the dispersed tDNAs and 5 S DNAs. Dispersed genes that appear to be derived from clusters of tandem repeats ("orphons") have been described for several gene families in invertebrates. The occurrence of this phenomenon in vertebrates as well, suggests that such dispersed genes may be a general feature of all eukaryotic genomes.

Characterization of two xenopus somatic 5S DNAs and one minor oocyte-specific 5S DNA.

The somatic 5S DNA from X. borealis (Xbs 5S DNA) and X. laevis (Xis 5S DNA) and a minor oocyte-specific 5S DNA from X. laevis (Xit 5S DNA) have been purified, and individual repeating units have been cloned and sequenced. The two somatic 5S DNAs differ from the major oocyte 5S DNAs in having GC-rich spacers, homogeneous repeat lengths and no "pseudogenes." The somatic 5S DNAs from the two species have similar spacer sequences with differences due to single base changes and insertions/deletions. The spacer of the minor oocyte-specific 5S DNA (Xit) has the AT-rich sequence characteristic of the major oocyte 5S DNAs from X. laevis and X. borealis, and contains one duplication that has diverged approximately 40%. Like the somatic 5S DNAs, Xit 5S DNA has a homogeneous length repeat and a unique nucleotide sequence in its spacer. The presence of variable-length spacer regions in a multigene family correlates with variables numbers of a simple sequence in the spacer regions.

Occlusion for successful class IV acid-etch restorations.

Properly placed acid-etch Class IV restorations may be dislodged due to occlusal interferences. When restoring an anterior tooth, the dentist should check the three regions of possible incisal prematurities: centric incisal contact, protrusive incisal contact, and midprotrusive incisal contact. By correcting potential problems in these regions before tooth restoration, the dentist helps to ensure longer lasting retention for the restoration.

DNA methylation patterns in the 5S DNAs of Xenopus laevis.

The frequency of cytosine methylation at specific sites in the somatic 5S DNA (X1s) and trace oocyte 5S DNA (X1t) of X. laevis has been determined using restriction enzymes that are inhibited by the presence of 5-methylcytosine (5mC) within their cleavage sequences. 5S DNA methylation patterns were determined in genomic DNA from mature red blood cells, which express neither type of 5S gene, and from liver, which expresses only X1s. All the sites examined in X1t are greater than 95% methylated in red cells and liver. In the X1s of red cells all the sites examined are methylated in greater than 95% of repeats, while in liver some sites are modified in only 90% of repeats. Repeats containing unmethylated sites are randomly distributed throughout the tandem arrays in both red cells and liver. The high levels of methylation for X1s are in marked contrast to the situation with other Xenopus genes which do have sites of significant undermethylation in tissues where they are active. Thus, undermethylation in active genetic regions may not be a general feature for all classes of eukaryotic genes.

Sequence heterogeneity of the ferripyoverdine uptake (fpvA), but not the ferric uptake regulator (fur), genes among strains of the fluorescent pseudomonads Pseudomonas aeruginosa, Pseudomonas aureofaciens, Pseudomonas fluorescens and Pseudomonas putida.

Pseudomonas aeruginosa, Pseudomonas aureofaciens, Pseudomonas fluorescens and Pseudomonas putida are of importance to medicine, agriculture and biocycling. These microbes acquire ferric ion via the use of the siderophores pyochelin and the family known as the pyoverdines or pseudobactins. The ferric uptake regulator (fur) gene is responsible, at least in part, for the regulation of siderophore synthesis and uptake in P. aeruginosa. To determine whether the organisms contain single or multiple homologues of the siderophore-related genes fpvA (ferripyoverdine uptake) and fur, and whether these homologues displayed sequence heterogeneity, their chromosomal DNAs were probed with fur and fpvA sequences. As a representative of a non-fluorescent pseudomonad, the bacterium Burkholderia (Pseudomonas) cepacia was also examined. The pseudomonads all contained fpvA- and fur-like homologues, and heterogeneity was observed among the different species. The presence of two or more fpvA-like genes is indicated in all of the fluorescent pseudomonads surveyed. In contrast, B. cepacia DNA either did not hybridize to these probes, or did so only very weakly, suggesting that fur- and fpvA-like homologues are either absent or significantly different in B. cepacia compared to the fluorescent pseudomonads examined.

Evolution of chick type I procollagen genes.

Although the major types of vertebrate collagen have a number of structural properties in common, significant DNA sequence homologies have not been detected between different portions of the helical coding domains within the same gene or between different genes. However, under non-stringen hybridization conditions we found considerable cross-homology within and between alpha 1(I) and alpha 2(I) chick cDNAs in the coding regions for helical sequences. Detailed analyses at the DNA sequence level have led us to propose that the gene for chick pro alpha 2(I) collagen arose from a 9-bp primordial sequence. A consensus sequence for the 9-bp repeat was derived: GGTCCTCCT, which codes for a Gly-Pro-Pro triplet. The primordial ancestor of this 9-bp unit, GGTCCTXCT, apparently underwent duplication and divergence. Each resulting 9-bp sequence was triplicated to form a 27-bp domain, and a condensation event produced a 54-bp domain. This genetic unit then underwent multiple rounds of amplification to form the ancestral gene for the full-length helical section of alpha 2(I). A different 9-bp consensus sequence (GGTCCCCCC) seems to have been the basis of the chick pro alpha 1(I) gene.

Practical means for the study of electron correlation in atoms.

Electron correlation is basic to the understanding of a diverse range of physical and chemical phenomena, yet, there have been no direct measurements of the correlated motion of electrons. Measurement of the correlated momenta of atomic electrons is possible via electron-impact double ionization provided that the ionizing collisions are both impulsive and binary, and the three-body scattering mechanism is known. The results reported here satisfy these conditions, and a practical means for the study of atomic electron correlation through measurement of two-electron momentum densities is presented.