Nucleotide Sequence Surrounding the Locus Marker D21S246 on Human Chromosome 21

Most ofthe human Not I linking clones identified to date areconsidered to be derived from CpG islands because ofthe recognitionsequence of this enzyme, and CpG islands have been reportedto be located around the 5' regions of genes. As a pilot study,we determined the complete nucleotide sequence (41,924 bp) ofa human cosmid clone (LL21NC02Q7A10) containing the marker D21S246originating from a Not I linking clone. As a result of sequenceanalysis, we successfully mapped and revealed the genomic genestructure for KIAA0002 previously reported as a cDNA clone.This gene consists of 15 exons and was shown to exist at theD21S246 locus on human chromosome 21q21.3–q22.1. Theseresults demonstrated that genomic marker-anchored DNA sequencingis a useful approach for the human genome project.     

A novel human type I hair keratin gene: evidence for two keratin hHa3 isoforms

We present the nucleotide and amino acid sequence for a novel human type I hair keratin, which could be identified through its high sequence homology and strict carboxyterminal length identity as a human ortholog of the murine hair keratin mHa3. Our hHa3 sequence differs, however, from that of a previously described hHa3 hair keratin (published only as an amino acid sequence; [13]) in 24 amino acid positions, 8 of which occur in the middle of the carboxyterminal domain. PCR of genomic DNA from 25 normal human subjects using a primer pair derived from sequence segments located in the 3-region of our hHa3 clone that encode conserved amino acid sequences in both keratins, resulted in the amplification of two distinct products of 0.38 kbp and 1.0 kbp. DNA sequence analysis of the cloned PCR products allowed identification of the 0.38 kb sequence as that originating from Yuet al. [13] and the 1.0 kb sequence as that being derived from our data. The difference in fragment length was due to unique intron 6 sequences, indicating that these two keratin species are encoded by genes of their own. Moreover, extensive Southern blot analyses with DNA from 25 unrelated individuals of different races using a 3-noncoding sequence from our keratin and the intron 6 sequence of the keratin of Yuet al. [13], as hybridization probes showed that both keratin genes are present as single copy sequences occurring ubiquitously and without gross alterations in the human genome. Collectively, these data demonstrate that the human type I hair keratin described in this paper represents an isoform of the previously described hHa3 keratin. We propose that these hHa3 isoforms be named in chronological order of discovery hHa3-I and hHa3-II.     

Restriction endonucleases from Selenomonas ruminantium which recognize and cleave 5′-AT/TAAT-3′

Two natural isolates from fallow-deer rumen identified as Selenomonas ruminantium were found to produce a restriction endonuclease which we called Sru4DI. This enzyme was isolated from cell extracts by phosphocellulose chromatography. Analysis of the Sru4DI recognition site showed that Sru4DI recognizes the hexanucleotide sequence 5-AT/TAAT-3 generating 5 dinucleotide protruding ends upon cleavage and thus is a true isoschizomer of VspI, a restriction enzyme isolated from Vibrio sp.     

Biochemical genetics of methylglyoxal dehydrogenases in the laboratory rat (Rattus norvegicus)

A genetic locus controlling the electrophoretic mobility of a methylglyoxal dehydrogenase (EC 1.2.1.23) in the rat is described. The locus, designatedMgd1, is expressed in liver and kidney. Inbred rat strains have fixed either alleleMgd1 ^a or alleleMgd1 ^b . Codominant expression is observed in heterozygotes, providing evidence for a tetrameric enzyme structure. Backcross progenies showed the expected 1:1 segregation ratio, and there is evidence thatMgd1 is linked toPep3 andFh1 on chromosome 13. There is also evidence for two additional methylglyoxal dehydrogenases:Mgd2, present in liver and kidney, andMgd3, present only in heart.Supported by the Deutsche Forschungsgemeinschaft (Grant Be 352/18-1).     

Investigations of mechanisms of resistance to bipyridyl herbicides in Arctotheca calendula (L.) Levyns

The mechanism of resistance to diquat and paraquat was investigated in a bipyridyl-herbicide-resistant biotype of Arctotheca calendula (L.) Levyns. No differences were observed in the interactions of these herbicides with Photo-system I, the active site, in thylakoids isolated from resistant and susceptible biotypes. Likewise, absorption of herbicide through the cuticle and gross translocation were identical in plants of the two biotypes. Foliar application of either 25 g ha⁻¹ diquat or 200 g ha⁻¹ paraquat rapidly inhibited CO₂-dependent O₂ evolution of leaf segments of the susceptible biotype. O₂ evolution of leaf segments of the resistant biotype was less affected by these treatments. Fluorescence imaging was used to observe visually, as fluorescence quenching, the penetration of herbicide to the active site. These experiments demonstrated that diquat appears at the active site more slowly in the resistant biotype compared to the susceptible biotype. HCO₃-dependent O₂ evolution of thin leaf slices was less inhibited by diquat in the resistant biotype than in the susceptible biotype. The mechanism of resistance to the bipyridyl herbicides in this biotype of A. calendula is not a result of changes at the active site, decreased herbicide absorption or decreased translocation, but appears to be due to reduced herbicide penetration to the active site.     

Activity of the tetramer and octamer of glutamine synthetase isoforms during primary leaf ontogeny of sugar beet (Beta vulgaris L.)

In extracts from the primary leaf blade of sugar beet (Beta vulgaris L.) we separated a chloroplastic isoform (GS 2) of glutamine synthetase (GS, EC 6.3.1.2) and one or two (depending on leaf age) cytosolic isoforms (GS 1a and GS 1b). The latter were prominent in the early (GS 1a) and late stages of leaf ontogeny (GS 1a and GS 1b), whereas during leaf maturation GS 2 was the predominantly active GS isoform. The GS 1 isoforms were active exclusively in the octameric state although tetrameric GS 1 protein was detected immunologically. Their activity stayed at a relatively constant level during leaf ontogeny; an increase was observed only in the senescent leaf. The activity of GS 2, however, changed drastically during primary leaf ontogeny and was modulated by changes in the oligomeric state of the active enzyme. In the early and late stages of leaf ontogeny when GS 2 activity was low (lower than that of the GS 1 isoforms), GS 2 was active only in the octameric state. In the maturing leaf, when GS 2 activity had reached its maximum level (much higher than that of the GS 1 isoforms), 80 of total GS 2 activity was due the activity of the tetrameric form of the enzyme and 20 was due to octameric GS 2. Tetrameric GS 2 was a hetero-tetramer and thus not the unspecific dissociation product of homo-octameric GS 2. In addition, GS 2 activity was modulated by an activation/inactivation of the tetrameric GS 2 protein. Due to an activation of the GS 2 tetramer, the activity of tetrameric GS 2 increased during leaf maturation from zero level 23-fold compared with that of GS 1a and 18-fold compared with that of GS 1b. Possible activators of tetrameric GS 2 are thiol-reactive substances. During leaf senescence, GS 2 activity decreased to zero; this decrease was due to an inactivation of the tetrameric GS 2 protein probably caused by oxidation.Abbreviations FLL final lamina length - FPLC fast protein liquid chromatography - GS glutamine synthetase - GHA -glutamyl hydroxamate - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase Dr. Roger Wallsgrove's (Rothamsted Experimental Station, Harpenden, UK) generous gift of GS antiserum is greatly appreciated.     

Growth hormone and insulin-like growth factor I concentrations in bulls of various growth hormone genotypes

A leucine/valine substitution at amino acid position 127 was identified by the polymerase chain reaction and restriction fragment length polymorphism in the bovine growth hormone gene. Genotyping was performed in 84 AI bulls of three different breeds, in which plasma concentrations of growth hormone (GH) and insulin-like growth factor I (IGF-1) were also measured. Gene frequencies of variants L (leucine) und V (valine) were 0.80/0.20 (Black and White), 0.90/0.10 (Brown), 0.71/0.29 (Simmental). Hormone concentrations were measured during different physiological conditions (normal feeding, fasting, realimentation) in the majority of animals. Generally, genotype LL was associated with higher concentrations of GH than LV. This difference was significant in Black and White bulls (P < 0.05). In contrast, IGF-1 concentrations were higher in LV than in LL animals. This was most pronounced in mature, realimented Simmental bulls. We conclude that the various GH alleles influence the circulating concentrations of GH and IGF-1.     

Electron microscopic structural analysis of Photosystem I,Photosystem II,and the cytochromeb6/f complex from green plants and cyanobacteria

Electron microscopy (EM) in combination with image analysis is a powerful technique to study protein structure at low- and high resolution. Since electron micrographs of biological objects are very noisy, substantial improvement of image quality can be obtained by averaging individual projections. Crystallographic and noncrystallographic averaging methods are available and have been applied to study projections of the large protein complexes embedded in photosynthetic membranes from cyanobacteria and higher plants. Results of EM on monomeric and trimeric Photosystem I complexes, on monomeric and dimeric Photosystem II complexes, and on the monomeric cytochromeb6/f complex are discussed.