DNA polymorphisms associated with a new α1-antitrypsin PI Q0 variant (PI Q0riedenburg)

Summary A new PI Q0 variant (PI Q0_riedenburg) is described; it is caused by a complete deletion of the ₁-antitrypsin (₁AT) gene. The deletion gives rise to four new restriction fragment length polymorphisms (RFLPs) detected with a genomic probe of the 5 region of the gene. Analysis of the RFLPs indicates that the deletion starts immediately upstream of exon Ic. The deletion extends into the 3 flanking region of the gene but does not include the ₁AT-related gene (the PIL gene), which is located 12 kb downstream of the ₁AT gene.     

Human αB-crystallin (CRYA2) gene mapped to chromosome 11q12-q23

Summary The B-crystallin gene (CRYA2) encodes the abundant lens protein B-crystallin. A panel of human/ rodent hybrid cell lines, derived from five different parental combinations, was characterized with respect to human chromosomal content and the presence of well-established human chromosome-specific markers. This panel was screened for the presence of CRYA2, using the third exon of the hamster B-crystallin gene as a probe. The patterns of segregation of CRYA2 with individual human chromosomes show the highest degree of concordance between CRYA2 and chromosome 11. Using cell hybrids containing translocated and/or partially deleted human chromosomes, the CRYA2 gene was localized to 11q₁₂-11q₂₃.     

Localization of the LDHA gene to 11p14→11p15 by in situ hybridization of an LDHA cDNA probe to two translocations with breakpoints in 11p13

Summary Lymphoblastoid cell lines established from two individuals with apparently balanced translocations involving 11p13 were used for LDHA regional localization. The karyotypes were 46,XY,t(4;11)(q21;p13) and 46,XY,t(1;11) (p22;p13). In situ hybridization of a human LDHA cDNA probe to chromosome preparations from these cell lines resulted in specific labeling over bands p14p15 of the normal chromosomes 11 and over bands 11p1411p15 of the derivative chromosomes 4 and 1. These results exclude LDHA from any region proximal to 11p13 and localize the gene to 11p1411p15.     

Evidence for wheat-rye nucleolar competition (amphiplasty) in triticale by silver-staining procedure

Summary Amphiplasty in hexaploid triticale, the artificial amphiploid of tetraploid wheat and diploid rye, is analyzed for the first time using a modified, highly reproducible, silver-staining procedure. A comparative analysis of metaphase somatic cells by phase contrast, C-banding and silver-staining of the hexaploid triticale cv. Cachirulo and its parents, namely, the tetraploid durum wheat cv. Enano de Andujar and the diploid rye cv. Petkus has been made. Two silver-stained nucleolar organizer regions (Ag-NORs) (the chromosome pair 1 R) are observed in all rye plants analyzed, whereas four Ag-NORs (chromosome pairs 1 B and 6 B) are found both in the tetraploid wheat parent and in the triticale. The rye Ag-NORs are absent in the triticale. Since the Agstaining reaction of NORs can be considered as an indication for genetic activity, the silver procedure can be used to visualize gene functionality at the rDNA sites with conventional light microscopy and, consequently, the modified Ag-staining method described can be very useful in analyzing the amphiplasty phenomenon in natural or artificial hybrid combinations and derivatives in the Triticum group and its relatives.     

Confirmation of assignment of the human α1-crystallin gene (CRYA1) to chromosome 21 with regional localization to q22.3

Summary The crystallins are highly conserved structural proteins universally found in the eye lens of all vertebrate species. In mammals, three immunologically distinct classes are present, -, -, and -crystallins, and each class represents a multigene family. The -crystallin gene family consists of 1-crystallin (CRYA1) and 2-crystallin (CRYA2) genes (previously designated A-and B-crystallin, respectively), which show extensive sequence homology. We constructed a synthetic oligonucleotide probe of 25 bases corresponding to a specific region of the human 1-crystallin gene sequence. This 25-mer probe bears little sequence homology to human 2-crystallin gene and does not cross-hybridize to 2-crystallin sequences in Southern blot analysis. Using this unique synthetic probe, we have demonstrated the identity of the 1-crystallin gene in human genomic DNA. In addition, we have also confirmed its chromosomal location on human chromosome 21. Finally, we have regionally localized the gene to q22.3 by using both Southern blot analysis of a panel of cell hybrids containing different parts of human chromosome 21, and in situ hybridization to metaphase chromosomes. The use of synthetic oligonucleotide probes specific for individual genes should be useful in identifying and mapping members of multigene families.     

Molecular organization of the human CD3 gene family on chromosome 11q23

The genes encoding three invariant components of the human T-cell antigen receptor, the CD3 , , and chains, are located on human chromosome 11 at band q23. We isolated cosmid clones containing the human CD3 and chain genes in vectors designed for rapid and efficient chromosome walking. The human CD3 gene was located in the region immediately downstream of the CD3 and genes using synthetic oligonucleotide probes and the localization of this gene confirmed by DNA sequencing. Detailed restriction mapping of the CD3 locus demonstrated that all three CD3 subunits are encoded within 60 kb of DNA with the CD3 gene located 26 kb downstream of the CD3 and genes. Analysis of genomic DNA on pulsed field gels using probes isolated from these cosmid clones defined a physical map of 750 kb spanning the CD3 locus on human chromosome 11g23. The CD3 genes thus comprise a multigene family encoding cell surface components important for transmembrane signaling on T lymphocytes. The arrangement of these genes suggest that they may share common regulatory elements for the control of gene expression during T-cell ontogeny.     

Enzymatic semisynthesis of aprotinin homologues mutated in P′ positions

The replacement of amino acids in the P₁ and P₂ position of aprotinin, the bovine pancreatic trypsin inhibitor, is described. Using the modified inhibitor as starting material, with the hydrolyzed reactive-site peptide bond Lys¹⁵-Ala¹⁶, the residues P₁ (Ala¹⁶) and P₂ (Arg¹⁷) were split off by the action of aminopeptidase K. Incorporation of suitable dipeptides containing a basic residue (Lys or Arg) in the C-terminal position was carried out in a one pot reaction involving trypsin-catalyzed coupling. In this way, the native fragment Ala¹⁶-Arg¹⁷ was reintroduced and also replaced by Gly-Arg, Ala-Lys, and Leu-Arg yielding intact inhibitor molecules. The mechanism for incorporation of dipeptides was investigated by treating the aprotinin derivative with the Arg¹⁷-Ile¹⁸ peptide bond hydrolyzed with trypsin under proteosynthetic conditions. We established that only inhibitor molecules cleaved between Lys¹⁵ and Xaa¹⁶ are intermediates leading to the desired products. The inhibitory properties of the new aprotinin homologues were tested, and the significance of the P₁ residue for the inhibition of trypsin, kallikrein, and chymotrypsin was deduced.     

ADP-ribosyltransferase activity in myelin membranes isolated from human brain

An ADP-ribosyltransferase has been identified in compact myelin and in several white matter fractions which contain less compact myelin, fractionated on the basis of increasing protein/lipid ratios. One fraction the P₃A contained the greatest activity although the activity in compact myelin was only slightly less. The ADP-ribosyltransferase activity of solubilized myelin was stimulated by increasing amounts of GTPS and was specific for the -isomer of NAD. Although ADP-ribosylation was demonstrated with the heterotrimeric G proteins in the 40–50 kDa range, the substrate for the ADP-ribosyltransferase in the 20 kDa range was identified as MBP. ADP-ribosyltransferase; myelin basic protein; signal transduction.Abbreviations ADP-ribose adenosine diphosphate ribose - APAD 3-acetylpyridine adenine dinucleotide - ATP adenosine triphosphate - C-1, 2, 3 etc MBP components isolated by CM52 chromatography - EDTA ethylenediaminetetraacetic acid - GTP guanosine triphosphate - GTPS guanosine 5-(3-0-thio)triphosphate - INH isonicotinic acid hydrazide - MBP myelin basic protein - NAD nicotinamide adenine dinucleotide - PMSF phenylmethylsulfonyl fluoride - PLP proteolipid protein Special issue dedicated to Dr. Leon S. Wolfe.     

The function of root systems in mineral nutrition of watercress (Rorippa nasturtium-aquaticum (L) Hayek)

Summary The ability of adventitious and basal root systems of watercress (Rorippa nasturtium-aquaticum (L) Hayek) to absorb mineral nutrients from surrounding media has been demonstrated using radioisotopes ³²P, ⁸⁶Rb and ⁵⁹Fe. Controlled experiments on single whole plants cultured in a dualmedium-apparatus, indicate that both root systems have a capacity for nutrient absorption. Analysis of axillary shoots formed during a seven day experimental period show that a greater proportion of phosphate and potassium, gained from the ambient media, was absorbed by the adventitious root system, although there was a greater mass of basal root tissue. Extensive translocation of nutrients to actively growing plant organs occurs from absorption sites on both root systems. re]19760504     

Quantitative electron probe microanalysis of leech photoreceptors

Summary The photoreceptive microvilli in the visual cells of the leech protrude into a large intracellular vacuole which is but an extracellular compartment (ionic composition unknown), because it communicates with the extracellular space by narrow ( 20 nm) clefts (septate junctions) of unknown permeability properties. Application of Thiéry's cytochemical silver proteinate method reveals that the vacuole contains carbohydrate-rich material. We used electron probe microanalysis of dry, ultrathin cryosections to determine quantitatively the elemental (K, Na, Cl, Mg, Ca, P, S) composition of the cytoplasm, vacuole and extracellular space.The composition of the vacuole is similar to that of the extracellular space, as shown by the comparable Na/K (11 to 13) and K/Ca (1.8 to 2.2) ratios in these two compartments. There are neglible concentration gradients for Na, K and Cl between vacuole and extracellular space. The vacuole has a high S content and a relatively large deficit of Cl compared to [Na]+[K]+2 [Ca]. Thus the data indicate that the vacuole is in ionic communication with the extracellular space and contains sulfonated glycoprotein(s) that can partially exclude Cl; electroneutrality is maintained in part by these organic anions. The cytoplasmic K concentration (393±30 mmol/kg dry wt) is comparable to that in other nerve cells. The cytoplasmic Cl concentration (216±14 mmol/kg dry wt) is relatively high: significantly (P<0.001) higher than the cytoplasmic Na (130±15 mmol/kg dry wt). The high cytoplasmic Cl content is in excess of that predicted by passive distribution, and suggests the operation of a Cl pump.     

Both common and specific genetic factors are involved in polygenic resistance of pepper to several potyviruses

Absolute resistance to potato virus Y pathotype 0 (PVY 0), potyvirus E and chili veinal mottle virus (CVMV) and a partial resistance to potato virus Y pathotype 1,2 (PVY 1,2) were found in an Indian pepper line, Perennial. In the doubled haploid (DH) progeny from the F₁ of a cross Perennial by Yolo Wonder, resistance to CVMV was confered by two independent genes, one with a clear dominant effect. Resistance to PVY and potyvirus E was quantitatively expressed and controlled by several recessive genetic factors. Genetic analysis showed that fewer resistance factors were necessary to explain resistance to PVY (0) and potyvirus E than resistance to PVY(1,2). Genetic correlations between resistances to the different potyviruses in the DH progeny showed that most of genetic factors involved in PVY(0) resistance appear to be also involved in potyvirus E resistance, and some of these polyvalent factors may be also involved in PVY(1,2) resistance but, in this case, additional specific genes were necessary. One of the two CVMV resistance genes seems to be implicated in potyvirus E resistance. Thus, the polygenic resistance of Perennial to these potyviruses was due both to polyvalent genetic factors, i.e. factors that apparently interact with several viruses, and strain-specific genetic factors.     

The addition of Dasypyrum villosum (L.) Candargy chromosomes to durum wheat (Triticum durum Desf.)

Summary Six monosomic addition lines were produced in which different Dasypyrum villosum (L.) Candargy chromosomes were added to the chromosome complement of Triticum durum Desf. cv. Creso. Each added alien chromosome was found to have a specific effect on plant morphology and fertility. Transmission rate varied widely (from 7.5 to 27.7%) among the six univalent chromosomes. Different monotelosomic addition plants derived by a relatively high frequency of chromosome misdivision were isolated. The addition lines should be useful for studying Dasypyrum chromosome homoeology and the introduction of alien variation into durum and common wheats.Research supported by a grant from the Italian Research Council for Finalized Project IPRA. Sub-project Plant Breeding, Paper No. 1095     

A new type of inversion of a human Y chromosome

Summary Using chromosome banding techniques, a phenotypically normal male was found to have an abnormal banding pattern of the Y chromosome. By the constitutive heterochromatin staining method, a darkly stained band was located on the short arm and the proximal region of the long arm. The quinacrine staining method also showed a similar abnormal banding pattern: a brightly fluorescing band was seen on the short arm and the proximal region of the long arm. By the conventional Giemsa staining method, however, no specific morphological abnormality was detected in the aberrant Y. On detailed karyotype analyses no recognizable abnormality of banding patterns of any other chromosome was found aside from the abnormal Y. The abnormality was determined to be a complex inversion of the Y chromosome, which is described as 46,X,inv(Y)(pterp11::q11q12::cen::q12qter).     

Silver staining the chromosome scaffold

Cytological silver-staining procedures reveal the presence of a core running along the chromatid axes of isolated HeLa mitotic chromosomes. In this communication we examine the relationship between this core and the nonhistone chromosome scaffolding, isolated and characterized in previous publications from this laboratory. When chromosomes on coverslips were subjected to the steps used for scaffold isolation in vitro and subsequently stained with silver, the characteristic core staining was unaffected. Control experiments suggested that the core does not contain large amounts of DNA. When scaffolds were isolated in vitro, centrifuged onto electron microscope grids, and stained with silver, they were found to stain selectively under conditions where specific core staining was observed in intact chromosomes. These results suggest that the nonhistone scaffolding is the principal target of the silver stain in chromosomes.     

High frequency of triplicated α-globin loci and absence or low frequency of α thalassemia in Polynesian Samoans

Summary Most of the population in certain areas of Melanesia have one -globin gene deletion ( thal₂). It is thought that the high frequencies of thal₂ in this population is due to a selective advantage given by malaria infection to carriers of thal₂. We are interested in neighboring Polynesia which, although adjacent to Melanesia, has always been free of malaria due to the absence of the vector anopheles. We studied 60 Polynesian Samoans and 150 Malaysians by restriction endonuclease gene mapping using Eco RI, Bam HI, and Bgl II and hybridization to ³²P-labeled -globin gene probe. Seven among the 60 (11.7%) Samoans had triplicated -globin loci type 1, while none had thal₂. On digestion with Bgl II the third -globin gene was found in an additional 3.7kb fragment in all seven Samoans with triplicated -globin loci, while digestion with Bam HI produced an abnormal elongated 18.2 kb fragment carrying -globin genes in addition to the normal 14.5 kb fragment. None of the Polynesian Samoans had thal₂ or thal₁. Only two of the Malaysians had triplicated -globin loci.     

Autoradiographic studies of the human Y chromosome

An autoradiographic analysis (using continuous labeling with tritiated thymidine) was made on 317 cells from four normal males. The labeling pattern of the Y chromosome was compared to the first and the last chromosomes to complete replication as well as to G21–22. The Y chromosome was never found to be the last chromosome in the cell to complete replication. Instead, it completed DNA synthesis relatively early (usually among the first 10 chromosomes) but had a distinctively heavy label during the earliest stages of late-S. In 51% of those cells with one labeled G+Y chromosome, a G21–22 was labeled and the Y was not.—It was concluded, therefore, that the human Y chromosome is not a late-replicating chromosome but terminates replication earlier than most of the autosomes. In addition, the Y chromosome cannot be distinguished from the G chromosomes on the basis of a consistent and differential labeling pattern.Supported by USPHS Grant GM 15361.     

Sequence analysis of 22 kDa-like α-coixin genes and their comparison with homologous zein and kafirin genes reveals highly conserved protein structure and regulatory elements

Several genomic and cDNA clones encoding the 22 kDa-like -coixin, the -prolamin of Coix seeds, were isolated and sequenced. Three contiguous 22 kDa-like -coixin genes designated -3A, -3B and -3C were found in the 15 kb -3 genomic clone. The -3A and -3C genes presented in-frame stop codons at position +652. The two genes with truncated ORFs are flanking the -3B gene, suggesting that the three -coixin genes may have arisen by tandem duplication and that the stop codon was introduced before the duplication.Comparison of the deduced amino acid sequences of -coixin clones with the published sequences of 22 kDa -zein and 22 kDa-like -kafirin revealed a highly conserved protein structure. The protein consists of an N-terminus, containing the signal peptide, followed by ten highly conserved tandem repeats of 15–20 amino acids flanked by polyglutamines, and a short C-terminus. The difference between the 22 kDa-like -prolamins and the 19 kDa -zein lies in the fact that the 19 kDa protein is exactly one repeat motif shorter than the 22 kDa proteins.Several putative regulatory sequences common to the zein and kafirin genes were identified within both the 5 and 3 flanking regions of -3B. Nucleotide sequences that match the consensus TATA, CATC and the ca. –300 prolamin box are present at conserved positions in -3B relative to zein and kafirin genes. Two putative Opaque-2 boxes are present in -3B that occupies approximately the same positions as those identified for the 22 kDa -zein and -kafirin genes. Southern hybridization, using a fragment of a maize Opaque-2 cDNA clone as a probe, confirmed the presence of Opaque-2 homologous sequences in the Coix and sorghum genomes.The overall results suggest that the structural and regulatory genes involved in the expression of the 22 kDa-like -prolamin genes of Coix, sorghum and maize, originated from a common ancestor, and that variations were introduced in the structural and regulatory sequences after species separation.     

Sequence- and Regio-Selectivity in the Montmorillonite-Catalyzed Synthesis of RNA

The six binary montmorillonite clay-catalyzed reactions of the5-phosphorimidazolides of adenosine, cytidine, guanosine anduridine were performed and the eight dimers from each reactionwere separated and analyzed by HPLC. A 16–51-fold higher yieldof the 5-purine-pyrimidine dimers over that of the5-pyrimidine-purines was observed. The total yield of the5-purine-pyrimidine dimers was in the 50–70% range while thatof the 5-pyrimidine-purine dimers was 1.3–7.0%. Less sequenceselectivity was observed in the homodimers formed.Regioselectivity for the formation of 3, 5-phosphodiesterbonds over that found in the absence of clay was observed. The5-purine-pyrimidine, 5-pyrimidine-pyrimidine and5-purine-purine dimers had 3, 5-links in about half of theirphosphodiester bonds. The percent phosphodiester links in the5-pyrimidine-pyrimidine dimers was 18%, a value close to thatobserved in the absence of the montmorillonite catalyst. Themontmorillonite-catalyzed reaction of all four activatednucleotides was performed and the 24 products were separated andanalyzed. The trends observed in the binary reactions wereconfirmed and the results also showed that the relativereactivity of the activated monomers was A>G>C>U in theratio 8.2: 4.8: 1.3: 1 respectively. No 5-pyrimidine-purineswith a 5-U and pG³pU, pC³pAand pC³pG weredetected. These studies suggest that a limited population ofRNAs would have formed in catalyzed prebiotic reactions.     

Steroid 5α-reductase Type 1 Immunolocalized in the Rat Peripheral Nervous System and Paraganglia

Steroid 5-reductase is an enzyme that converts a number of steroids with a C-4, 5 double bond and C-3 ketone to 5- reduced metabolites. This enzyme has been suggested to play a role in brain development and myelination in the rat nervous system. In the present study, we examined the cellular and subcellular localization of the enzyme immunocytochemically in the rat peripheral nervous system and paraganglia using a polyclonal antibody against rat 5-reductase type 1. Light and electron microscopical studies localized 5-reductase in the Schwann cells of myelinated and unmyelinated nerve fibres, the satellite cells of the ganglia, the enteric glial cells and the supporting/sustentacular cells of the paraganglia. In the myelinated nerve fibres, immunoreactivity was observed in the outer loops, the nodes of Ranvier and the Schmidt–Lanterman incisures. Subcellularly, the immunoreactivity was localized in the cytopl asm of various glial cells. No immunoreactivity was observed in the myelin membrane, the axon or the neuronal perikaryon. These findings suggest that 5-reductase is widely distributed in glial cells, and that, in addition to myelination, 5-reduced steroids play a role in some glial functions in the peripheral nervous system.     

Efficient determination of angles subtended by Cα-Hα and N-HN vectors in proteins via dipole-dipole cross-correlation§

The angle CH,NHN subtended by the internuclear vectors 13C-H and 15N-HN in doubly-labeled proteins can be determined by observing the effect of cross-correlation between the dipolar interactions on zero- and double-quantum coherences involving 13C and 15N. Two complementary 2D experiments with the appearance of 15N-HN correlation spectra yield signal intensities that depend on the rate of interconversion through cross-correlated relaxation of in-phase and doubly antiphase zero- and double-quantum coherences. The ratio of the signal intensities in the two experiments bears a simple relationship to the cross-correlation rate, and hence to the angle CH,NHN. Assuming planarity of the peptide bond, the dihedral angle (between C and C) can be determined from the knowledge of CH,NHN. The experiments are very time-effective and provide good sensitivity and excellent spectral resolution.