The role of cytochemistry in human genetic research

The role of cytochemistry in human genetics is reviewed. In basic research, autoradiography and cytochemical staining procedures for DNA, RNA, proteins and other cell constituents have contributed to the understanding of the way DNA is localized, duplicated and translated. The development of new "banding techniques" for the identification of human chromosomes and parts of these together with somatic cell hybridization procedures have significantly contributed to the mapping of the human genome. Cytochemical methods have also been of great help in the elucidation of the responsible molecular defects in Mendelian disorders based on a single gene mutation. The combination of immunological methods and electron-microscopical cytochemistry now enables different posttranslational processing defects to be related to various subcellular compartments. Cytochemistry is also likely to be of importance for the direct demonstration of gene mutations using recombinant DNA technology. Examples are given of contributions of cytochemical methods to the early diagnosis and prevention of congenital disorders. The main examples are the early diagnosis of patients with a chromosomal aberration and of carriers with a balanced translocation. Early genetic counseling of couples at risk forms the basis for prevention of subsequent affected children. Cytochemistry also contributes to the early detection of heterozygotes of X-linked mutations. Finally, autoradiography and ultramicrochemical procedures have been of great help in improving the prenatal diagnosis of genetic metabolic diseases.     

Characterization of human chromosomal unit fibers

A structural component of mitotic chromosomes that partially explains the compaction of DNA within mitotic chromosomes is suggested on the basis of the occurrence of long, regular cylindrical structures in preparations of isolated human chromosomes. These structures, unit fibers, of a rather constant diameter of about 4,000 Å have been postulated to be formed by coiling of the 250T2–300 Å solenoid chromatin fiber that itself is formed by coiling of the 100 Å string of nucleosome fiber. The human chromatid would thus be composed by a hierarchy of helices with contraction ratios for DNA at each level of coiling of 7 (string of nucleosomes), 5 (solenoid) and 40 (4,000 Å unit fiber or super-solenoid) which results in an overall contraction ratio for DNA in the unit fiber structures of about 1,400, which is approximately 5-fold less than the final contraction of DNA in intact chromatids of condensed metaphase chromosomes. The present report concerns more detailed studies with respect to the dimensions and cytochemical properties of the unit fiber structures observed in preparations of isolated human mitotic chromosomes that provide direct and indirect evidence in support of their super-solenoid structure and relate to known properties of human mitotic chromosomes.     

A quantitative cytochemical assay of β-galactosidase in single cultured human skin fibroblasts

Summary A quantitative cytochemical method for the measurement of -galactosidase activity in cultured human skin fibroblasts has been developed using 5-bromo-4-chloro-3-indolyl--D-galactopyranoside as the indigogenic substrate. The method relies upon the oxidation of the primary reaction product by ferro/ferricyanide during which an insoluble indigo dye is generated as the final reaction product. The reaction was linear with time up to 60 min using the final cytochemical standard procedure. The enzyme showed maximum activity at pH 4.0 to 4.1. The concentration optima of indigogenic substrate and potassium ferro/ferricyanide were 3.67 mM and 3.13 mM respectively. The presence of sodium chloride activated -galactosidase up to 100 mM, but was inhibitory above that concentration. The enzyme was inhibited by N-ethylmaleimide, N-acetyl-D-galactosamine and heparin. The enzyme molecules were shown to diffuse out of the cells using media without a suitable inert colloid stabilizer. However, diffusion was completely prevented by using polyvinyl alcohol (PVA) grade G18/140. Air-drying of cells was essential to make the cell membrane permeabel to the substrate and, thereby, to avoid a pronounced lag phase. However, in a biochemical analysis, air-drying itself caused a decrease in enzyme activity to 43% of the control. Even after air-drying lysosomal latency could still be demonstrated by using PVA grade G04/140.Control persons, one carrier of and two patients with -galactosidase deficiency were easily identified as belonging to three separate groups by using the cytochemical assay.It is proposed that the quantitative cytochemical approach may also be applied to cultured human amniotic fluid cells or chorion biopsies giving a rapid prenatal diagnosis of -galactosidase deficiency due to the small number of cells needed in the analysis.     

Development of crystalline inclusions (“ergosterol crystals”) inNeurospora crassa

Summary Development of crystalline inclusions (ergosterol crystals) in snowflake, a morphological mutant ofNeurospora crassa has been examined. The inclusions which arise in membranebound organelles appear as electron dense deposits, increase in size, and occupy nearly all the space within the organelle at maturity. The presence of catalase activity in the organelle was not detected using cytochemical procedures employing diaminobenzidine.     

DNA microextraction from dried blood spots on filter paper blotters: potential applications to newborn screening

Summary Microextraction of DNA from dried blood specimens would ease specimen transport to centralized laboratory facilities for recombinant DNA diagnosis in the same manner as use of dried blood spots allowed the broad application of screening tests to newborn populations. A method is described which reproducibly yields 0.5g DNA from the dried equivalent of 50l whole blood. Though DNA yields decreased with storage of dried specimens at room temperature, good-quality DNA was still obtained. Sufficient DNA was routinely obtained for Southern blot analysis using repetitive and unique sequences. This microextraction procedure will allow immediate application of molecular genetic technology to direct newborn screening follow-up of disorders amenable to DNA diagnosis, such as sickle cell anemia, and may eventually permit primary DNA screening for specific mutations.     

Host-dependent modification of bacteriophage T7 and SAMase-negative T3 derivatives affecting their adsorption ability

Summary When passaging phage T7 and SAMase-negative T3 mutants betweenE. coli strains with identical (EcoB) or without (EcoO) DNA host specificity, phenotypically a host-controlled modification and restriction is observed. This phenomenon is not due to classical modification and restriction of the bacteriophage DNA but depends on the reversibly altered adsorption capacity of the phages on the different host strains.     

Comparison of human and chimpanzee ξ1 blobin genes

Summary The DNA base sequences of the entire chimpanzee 1 globin gene and an additional 1 kb of DNA flanking both the human and chimpanzee genes have been determined. Whereas the human 1 gene contains a termination codon in the sixth position, the chimpanzee gene appears to be functional. This finding confirms Proudfoot et al.'s suggestion that the human 1 gene was recently inactivated. Like the corresponding human 1 and 2 genes, the first and second introns of the chimpanzee 1 gene are occupied largely by tandem repeats of short oligonucleotides. These tandem repeats have undergone several rearrangements since the divergence of the human and chimpanzee 1 genes.     

Unstable visible mutations induced in Drosophila melanogaster by injections of oncogenic virus DNA into the polar plasm of early embryos

Summary When RSV DNA cloned in pBR 322 or DNA of simian adenovirus Sa7 (C8) is injected into the pole plasm of embryos of various Drosophila stocks, the progeny of 1–70% of the surviving flies display visible mutations. The mutagenesis is partially directed: the loci mutating due to retrovirus and adenovirus DNA do not everlap. The majority of resulting mutants are characterised by high instability: reversions and new mutations occur in them, which sometimes spread over the whole population(explosive instability). The injected sequences are revealed by dot-hybridization in the DNA of many mutant strains, but only rarely by Southern blotting procedures. The results show that the microinjection of oncovirus DNA into embryos is an approach for obtaining highly unstable strains even from wildtype stable Drosophila stocks without crosses with MR lines or the introduction of P elements. The sets of unstable mutations induced by oncovirus DNA is different from those in hybrid dysgenesis.     

Intranuclear differences in the response of Purkinje cell DNA of the rat cerebellum to bleomycin

Summary A single dose of the DNA-binding cytostatic agent bleomycin (100 g/g body weight, subcutaneously) was given to 10-day-old rats to study unscheduled repair DNA synthesis in nucleolar and in bulk nuclear chromatin of postmitotic Purkinje neurons. The Feulgen reaction and Hoechst 33342 staining were used for quantitative evaluation of nuclear DNA content and chromatin structure. The repair synthesis of DNA was detected by ³H-thymidine autoradiography.The data showed a lesser staining of Purkinje as well as granule cell DNA by Hoechst 33342 in bleomycin-treated animals than in controls, but there was no difference in staining with the Feulgen reation. The mechanisms of DNA staining by both cytochemical methods suggest that bleomycin reacted preferentially with AT-rich and single stranded DNA in cerebellar cells in vivo. Weak ³H-thymidine labelling was found in Purkinje cells of both control and treated rats, but in the latter group the labelling was more pronounced near or over the nucleolus. The enhanced unscheduled DNA synthesis in the nucleolar region of Purkinje cells of treated animals may be due to greater damage of DNA in this region or may indicate a greater ability of the nucleolar chromatin to repair its DNA.Dedicated to Professor Dr. Z. Lojda, Dr. Sc., on the occasion of his 60th birthday.     

Cytochemical contributions to differentiating GERL from the Golgi apparatus

Synopsis Recent studies from our laboratory are described which deal with endocrine cells (insulinoma, -cells of the pancreas, thyroid epithelial cells), pancreatic exocrine cells, and hepatocytes. These emphasize the importance of the hydrolase-rich specialized region of endoplasmic reticulum, known as GERL, in secretory cells. Also reviewed in this paper are the varied molecular transformations which apparently occur in GERL in different cell types, as reported from other laboratories as well as our own. Evidence of the continuity of GERL with rough endoplasmic reticulum is presented. Two hydrolytic enzyme activities in GERL, in addition to acid phosphatase activity, are recorded. Finally, the use of cytochemical staining procedures in the study of microperoxisomes is briefly described. The Histochemical Journal lecture 1976. Delivered to the Histochemistry and Cytochemistry Section of the Royal Microscopical Society on 14 September 1976     

Introns and higher-order structure in the evolution of serpins

Summary The serpins are a large family of eukaryotic proteins, many but not all of whose members are proteinase inhibitors. Most members of this family show relatively low sequence identity, but crystal structures determined for 6 different serpins are closely similar. The intron positions of 11 serpins, and the intron sizes in 9 of these 11, have been determined. There is considerable diversity in number, position, and size of introns among these serpins, though subsets show clear similarity or identity. Dendrograms derived from comparisons of DNA and amino acid sequences and of intron positions for the 11 serpins differ from each other and from dendrograms previously derived from protein sequences. These dendrograms are difficult to reconcile exclusively with a loss of introns from a large primordial set during the evolution of the serpin family. The tertiary structure of the serpins does support the idea that this protein family arose from an early recombination event which fused the amino and carboxyl domains. The structure of the carboxyl domain also suggests that an insertion subsequent to the fusion event contributed two strands of -sheet, which complemented three -sheet strands of the amino domain, to complete -sheet A, which is the central secondary structure feature of the serpins. Few of the introns lie between regions of secondary or tertiary structure, and it seems more likely that many were acquired subsequent to the early events of serpin evolution and have undergone multiple insertions, deletions, and migrations since, subject to the constraint of the serpin structure.Abbreviations Api -1-proteinase inhibitor (human) - Aci -1-antichymotrypsin (human) - Agt angiotensinogen (rat) - Oah ovalbumin (chicken) - Gyh gene y (chicken) - At3 antithrombin 3 (human) - Pi1 plasminogen activator inhibitor 1—endothelial (human) - Pi2 plasminogen activator inhibitor 2—placental (human) - Cli Cl inhibitor (human) - Apl antiplasmin (human) - Bz4 Z protein (barley).     

Cellular immunity in an annelid (Nereis diversicolor,Polychaeta): production of melanin by a subpopulation of granulocytes

Summary We attempted to identify the nature and origin of the pigment produced by the marine worm Nereis diversicolor in order to isolate, in inert brown capsules, foreign objects introduced into its body cavity. This brown pigment, characterized by cytochemical techniques, could be a melanin. The activity of the enzyme phenoloxidase responsible for melanin biosynthesis was detected by enzyme cytochemistry techniques in vacuoles and the Golgi apparatus of coelomocytes activated by the presence of foreign bodies. Morphological techniques combined with a monoclonal immunological probe enabled us to establish that the G₂ granulocytes contain both the precursor of the pigment in dense bodies and the capacity for phenoloxidase synthesis when activated to encapsulate foreign bodies. The G₂ granulocyte may therefore be compared to a melanocyte in which melanin is not stored as in mammals, but immediately extruded following synthesis in the form of a thick fluid.Abbreviations ACTH adrenocorticotropic hormone - Dopa L-3,4 dihydroxyphenylalanine - FITC fluorescein isothiocyanate - G ₁, G ₂, G ₃ granulocyte of types 1, 2, 3 - MSH melanocyte-stimulating hormone - proPo prophenoloxidase     

Identification of living spermatogenic cells of the mouse by transillumination-phase contrast microscopic technique for ‘in situ’ analyses of DNA polymerase activities

Summary The stages of spermatogenesis can be identified in freshly isolated, unstained adult mouse seminiferous tubules using a transillumination method. Late acrosome- and maturation phase spermatids, arranged in bundles at stages XII–VI give rise to a spotty transillumination pattern. Before spermiation, these cells form a continuous layer on the top of the seminiferous epithelium, recognized by a strong homogeneous central light absorption in the freshly isolated seminiferous tubules at stages VII and VIII. Other stages have a pale light absorption pattern. The accurate determination of the developmental stages of the germ cells was based on the morphology of the developing acrosomic system and of the nuclei of the spermatids, as revealed by phase contrast microscopy. Using this procedure, the activity levels of DNA polymerases and have been studied by autoradiography of squash preparations. Using endogenous templates, assay conditions that differentiate between the solubilized DNA polymerases and in vitro, were used to distinguish between these activities in situ in different stages of mouse spermatogenesis. Except in very late spermatids shortly before spermiation, DNA polymerases and were detectable in all cell types examined. Coinciding with the nuclear protein transitions, elongating spermatids at steps 10–12 and maturation phase spermatids at steps 13–14 showed high DNA polymerase activities. As no replication occurs in these cells, the observations support the view that both DNA polymerases and could be involved in repair DNA synthesis.     

Tentoxin effects onSorghum: The role of polyphenol oxidase

Summary In an ultrastructural and cytochemical study of tentoxin-treatedSorghum bicolor (L.) Moench, both bundle sheath and mesophyll plastids were severely affected, Plastids from chlorotic leaf areas lacked most internal membranes yet had plastid ribosomes and large fibrillar areas of plastid DNA. In recovered areas (mottled yellow and green), cells were found that had plastids of near-normal ultrastructure as well as the severely affected plastid-types found in chlorotic leaf areas. Polyphenol oxidase (PPO) cytochemistry of these mottled leaf areas indicated that all recovered mesophyll plastids had PPO whereas all the abnormal mesophyll plastids showed no activity. Because bundle sheath plastids ofSorghum have no PPO activity at any developmental stage, yet are affected by tentoxin, PPO cannot be uniquely affected by this toxin. We suggest that tentoxin may affect the transport of cytosolic proteins into the plastid.     

A new dynamic model system for the study of capture reactions for diffusable compounds in cytochemistry. II. Effect of the composition of the incubation medium on the trapping of phosphate ions in acid phosphatase cytochemistry

Synopsis A model system developed for the study of the dynamics of capture reactions for diffusable compounds in cytochemistry served as a basis for the experiments reported in the present paper. The model was used to study the effect of the composition of the cytochemical medium on the trapping of phosphate ions by lead (II) ions in acid phosphatase cytochemistry. In this system a phosphate-containing solution and a lead-containing solution (cytochemical medium) are pumped along opposite sides of a polyacrylamide film. The phosphate concentration at which measurable precipitation starts in the film (critical phosphate concentration) was taken as a measure of the trapping efficiency of the cytochemical medium. The addition of -glycerophosphate and cytidine-5-monophosphate to a buffered lead-containing solution resulted in a higher critical phosphate concentration. Both substrates had an effect on the crystal form of lead phosphate. The addition of chloride ions and acetone, as well as decreasing the molarity of the acetate buffer of the cytochemical medium, were found to lower the critical phosphate concentration, whereas the addition of fluoride ions, glucose, and sucrose had no effect. From the effect of variations in the composition of the cytochemical medium on the trapping efficiency and the turnover number of acid phosphatase in the medium, it was possible to predict which cytochemical medium would be the most suitable for the demonstration of acid phosphatase activity in guinea-pig peritoneal exudate cells. The results were in accordance with the localization of acid phosphatase activity: the higher the trapping efficiency and the turnover number, the higher the amount of precipitate and the number of positive enzymatic sites. In this way an improved cytochemical medium for acid phosphatase was developed.     

Replication of chromosomal DNA in diploid Drosophila melanogaster cells cultured in vitro

Replication rate and replicon sizes in chromosomal DNA of in vitro cultured diploid D. melanogaster cells were determined using autoradiography of ³H-thymidine labeled DNA. Synthesis of DNA in euchromatic and heterochromatic regions of Drosophila diploid cells occurs at different periods of the S phase which lasts 10 h. During the first 4 h the synthesis is observed only in euchromatic regions. The heterochromatic synthesis starts shortly before the synthesis in euchromatic regions is completed and lasts for 6 h until the end of the S phase. The cells were synchronized by 5fluorodeoxyuridine which blocked the diploid cell DNA synthesis. Synthesis was found to start simultaneously in most euchromatic replicons. In the majority of the replicons the synthesis started at a single point and proceeded bidirectionally. The average rate of DNA synthesis per fork was 12.5 m/h (38 kb). The mean distance between the middle points of adjacent labeled regions was 70 m (210 kb). The size of most replicons ranged from 40 to 120 m. — These estimates do not apply to the heterochromatic portions of the D. melanogaster genome since the measurements have been carried out on DNA preparations obtained during the first 2 h of the S phase. — On the average, a replicon can consist of 7 chromomeres since the size of a replicon in diploid cell chromosomal DNA and DNA length of a polytene chromomere average 210 and 30 kb, respectively.     

Assignment of the gene encoding the catalytic subunit Cβ of CAMP-dependent protein kinase to the p36 band on chromosome

Summary A cDNA for the human catalytic subunit (C) of cAMP-dependent protein kinase (PKA) has been cloned from a testis cDNA library. In the present study, we have determined the chromosomal localization of this gene using a cDNA for C as a probe. Southern blot analysis of genomic DNA from human/mouse cell hybrids revealed that the presence or absence of a 20-kbXbaI fragment, which hybridized with the C probe, was concordant with the presence of human chromosome 1.In situ hybridization to metaphase chromosome confirmed the somatic cell hybrid data and regionally mapped the C gene of PKA to the p36 band on chromosome 1.     

Differentiation of somatic mitochondria and the structural changes in mtDNA during development of the dicyemid <Emphasis Type="Italic">Dicyema japonicum</Emphasis> (Mesozoa)

Dicyemids (Mesozoa) are extremely simple multicellular parasites found in the kidneys of cephalopods. Their mitochondria are known to contain single-gene minicircle DNAs. However, it is not known if the minicircles represent the sole form of mitochondrial genome in these organisms. Here we demonstrate that high-molecular-weight (HMW) mtDNA is present in dicyemids. This form of mtDNA is probably limited to germ cells, and has been analyzed by PCR and Southern hybridization. In situ hybridization revealed that mtDNA is initially amplified during early embryogenesis, and then gradually decreases in copy number as larval development proceeds. Furthermore, we demonstrated using BrdU as a tracer that many of the mitochondria in terminally differentiated somatic cells no longer support DNA synthesis. Taking these observations into account, we propose an amplification-dilution model for mesozoan mtDNA. Stem mitochondria in the germ cells (1) amplify the HMW form of mtDNA in early embryos, followed by minicircle formation via DNA rearrangement, or (2) selectively replicate minicircles from the HMW DNA, concomitantly with the differentiation of the soma. Minicircle formation may itself lead to the loss of replication origins. Thereafter, the minicircles are simply distributed to daughter mitochondria without replication, resulting in the somatic mitochondria, which have lost the replicative form of the HMW mtDNA. The change in mtDNA configuration is discussed in relation to mitochondrial differentiation.