Transcription of the immunoglobulin kappa light-chain genes depends on the presence of a TATA box upstream of the leader gene segment and is regulated by an enhancer sequence in the large intron. In studying a rearranged mouse kappa light-chain gene we have now found that sequences between--90 and--160 base pairs (bp) upstream of the coding region are essential for correct transcription in gene transfer experiments. This region contains the deca- and pentadecanucleotide sequences TNATTTGCAT and TGCAGCCTGTGNCCAG, which we call dc and pd, respectively. Sequences related to dc and pd were found upstream of all human and mouse kappa-chain variable region (Vk) genes, upstream of lambda-chain variable region (V lambda) genes, and within the mouse heavy-chain enhancer. An inverted and complementary form of the dc element (ATGCAAATNA, called cd) occurs upstream of all heavy-chain variable region (VH) genes. The newly defined sequences may be involved in the control of immunoglobulin gene transcription.

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By mild treatment of tRNA;:& with acid (pH 2.9, 37", 3-4 hours) the base Y' , which according to RajBhandary, Khorana et al. is located right next to the anticodon, can be excised without breaking the chain. Y+ and the acid treated tRNAPhe (= tRNA&E) were characterized and the conversion of tRNAPhe to tRNAP,h,9 was investigated in detail.1. Yf is a rather hydrophobic compound of unknown structure. Y+ and ribose were found when the nucleoside Y was treated with acid. Some physical and chemical properties of Yf are reported. Yf can be isolated easily in pure form from oligonucleotides, tRNA:,h,e,,, or even unfractionated tRNAyeast (not tRNA,,l() by mild acid treatment and subsequent extraction into chloroform.2. tRNAgEl and tRNAPhe have very similar molecular sizes and shapes since their Sephadex elution profiles were overlapping. The facile separation of tRNAPhe and tRNA&El by countercurrent distribution, on columns of methylated albumin on Kieselgur or benzoylated DEAEcellulose is due, a t least in part, to the removal of a hydrophobic residue from an exposed region of the tRNAPhe.3. tRNA&g can be charged with Phe to the same extent as tRNAPhe. Since also the Michaelis constant of phenylalanyl tRNA synthetase is the same for both tRNAs (1.5 x lo-' M) and the maximal velocity of charging is decreased by only one third in tRNAGE, it can be concluded, that Y and probably the whole anticodon loop are not essential parts of the synthetase recognition region of tRNAPhe in homologous charging reactions. 4.It was found that tRNAgE and tRNAPhe from which the -CpCpA endgroups had been removed do not differ in their acceptor capacities for CTP and ATP.5 . I n the ribosome systems, Phe-tRNAP,h,", does not bind to poly (U) or other polynucleotides and does not transfer its amino acid onto growing peptide chains. The complete loss of codon recognition is attributed largely to a change in the conformation of the anticodon loop. The ultraviolet spectra of an Y-containing hexanucleotide and of its acid conversion products are in agreement with the notion of a strong interaction between Y+ and its neighbouring bases, as are the rate differences in the liberation of Yf from Y 111.5 times faster] and the hexanucleotide, respectively.6. The rate of tRNAEz formation, as measured by methylated albumin on Kieselgur-column chromatography and binding assays, is the same as the rate of Y+-liberation from the hexanucleotide. The acid induced conversion of tRNAPhe to tRNAP,",", is a completely selective reaction affecting Y as the only nucleoside of tRNAPhe.

The question of which germ-line V kappa genes are expressed was studied by sequencing 70 different cDNA clones from a human spleen library and one clone from a fetal liver library. The sequences were compared to a data base containing all germ-line V kappa gene and pseudogene sequences. In addition, 51 rearranged genomic V kappa genes, 170 cDNA and 74 kappa proteins from the literature were assigned to specific germ-line V kappa genes and included in the comparisons. Not all the known, potentially functional V kappa genes were found to be expressed, while some genes with minor defects are. The total number of expressed genes is smaller than expected: so far 21 germ-line genes and 5 pairs of duplicated identical genes are known to be transcribed. The corresponding numbers for rearranged genomic V kappa genes and kappa proteins are 17 plus 4 and 7 plus 7, respectively. A second aim of the study was to find out whether the expressed repertoire contains derivatives of germ-line V kappa genes still missing in our data base; no evidence for the existence of such genes was found. Several cDNA clones contained additional nucleotides between the V kappa and J kappa gene segments, which may be germ-line derived, inserted by terminal deoxynucleotidyl transferase or introduced by other mechanisms. Somatic gene conversion seems not to play a major role in creating the human kappa gene diversity. Various aspects of the hypermutation of kappa genes are discussed and the formation of block mutations, i.e. the alterations of two or more adjacent nucleotides is stressed as a remarkable feature of the process.

In this report 118 mouse V O genes are described which, together with the 22 V O genes reported previously (T. Kirschbaum et al., Eur. J. Immunol. 1998. 28: 1458-1466 amount to 140 genes that had been cloned and sequenced in our laboratory. For 73 of them cDNAs are known, i. e. they have to be considered functional genes, although 10 genes of this group have 1-bp deviations from the canonical promoter, splice site or heptanucleotide recombination signal sequences. Twenty V O genes have been defined as only potentially functional since they do not contain any defect, but no cDNAs have been found (yet) for them. Of the 140 V O genes 47 are pseudogenes. There are indications that two to five V O genes or pseudogenes exist in the O locus which we have not yet been able to clone. The 140 V O genes and pseudogenes were assigned to 18 gene families, 4 of them being one-member families. This differs from previous enumerations of the families only by the combination of the V O 9 and V O 10 families and by the addition of the V O dv gene as a new separate family. Sequence identity usually was 80 % or above within the gene families and 55-80 % between genes of different families. Many of the mouse V O gene families show significant homologies to the human ones, indicating that in evolution V O gene diversification predated the divergence of the primate and rodent clades.

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The long-range periodicity of mouse satellite DNA has been analyzed by digestion with five restriction nucleases. With all nucleases tested, a major repeat unit containing approximately 245 nucleotide pairs became apparent. Minor registers of shorter length were also detected. The total number of cleavage sites per haploid genome for each restriction enzyme as well as their positions relative to each other were determined. While endo R.EcoRII was known to cleave all of the satellite DNA, the other four restriction enzymes were found to generate only weak degradation patterns. The results taken together with quantitative analyses of codigestion experiments indicate that the recognition sequences for each of these four nucleases are clustered on separate parts of the satellite DNA. It is concluded that the satellite DNA, which appears homogeneous by digestion with endo R.EcoRI1, contains distinct segments each susceptible to degradation with one of the other nucleases. These results have certain implications for theories on the evolution of mouse satellite DNA. A simple mechanism of multiplication and divergence by mutation is not sufficient to explain the data. Additional and alternative processes which are relevant to the evolutionary considerations are &cussed.Simple sequence DNAs have been found in every eukaryotic organism investigated so far (for summaries see [1,2]). Despite their ubiquity and their relative abundance in many organisms it is unknown at present what functions they serve within the eukaryotic genome. It seems clear, however, that they are not transcribed into RNA. It is interesting that closely related species have quite different simple sequence DNAs. This phenomenon requires rapid generation and elimination of such sequences in the course of evolution by mechanisms which are currently not understood. Information pertaining to such genome processes will aid our knowledge of the evolution of eukaryotic organisms in general.In many cases, simple sequence DNAs can be separated from the rest of the DNA by isopycnic gradient centrifugation and are then called satellite DNAs. All satellite DNAs are repetitious in nature, yet they vary in their degree of homogeneity. One extreme class consists of exact repeats of a short oligonucleotide sequence the structure of which could be elucidated by sequence analyses satellite DNAs, notably those from mouse and guinea pig, has a more complex sequence composition [7,8]. Even though they also seem to consist of tandem repeats, the sequence of each of the repeats has been conserved to a much lesser degree, and the satellite therefore has a more divergent appearance.A novel approach has complemented the preexisting ones in the analysis of satellite DNAs, i.e. digestion with restriction nucleases. This technique has revealed highly regular long-range repeats superimposed on the short-range divergence patterns [9-151. Our previous studies have shown that mouse satellite DNA and guinea pig satellite I11 show such periodicities in digests with endo R . Hind11 and e...

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